Sialic acid glycosides, antigens, immunoadsorbents, and methods for their preparation

ABSTRACT

The stereochemistry of sialylation of an acceptor saccharide to obtain an α (2-3) or α (2-6) linkage is controlled to favor the α anomer by use of an aromatic ester of the sialyl reagent. The resulting intermediate α (2-3) and α (2-6) sialylated intermediate disaccharide blocks are useful in the synthesis of antigenic substances which can be used to raise antibodies useful in diagnosis and therapy, and can themselves be used as reagents in various applications. The preparation of the tetrasaccharide antigens corresponding to the 19-9 and sialyl-X antigens characteristic of malignant tissue illustrates the application of this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 07/545,999, filed Jun.28, 1990, now U.S. Pat. No. 5,296,594 which, in turn is a division ofU.S. Ser. No. 07/127,905, now U.S. Pat. No. 5,079,353, filed Dec. 2,1987, both of which are incorporated herein by reference in theirentirety.

Technical Field

The invention relates to the fields of oligosaccharide synthesis andantigen/antibody interaction. Specifically, the invention concernssynthesis of oligosaccharide haptens. These haptens can be used asantigens which are rendered immunogenic by conjugation to raiseantibodies useful in diagnosis and therapy. The synthetic haptens canalso be used in immunosorbents for preparation, isolation, removal andpurification of the corresponding antibodies, and are useful in assayreagents.

BACKGROUND OF THE INVENTION

The following references are cited in this Background Section:

1. Schauer, R., Adv Carbohydr Chem Biochem (1982) 40:131 to 234.

2. Corfield, A. P., et al, "Sialic Acids, Chemistry, Metabolism andFunction", pp 5-50, Schauer, R., ed., Springer-Verlag, New York (1982).

3. Hakomori, S., TIBS (1984) 453 to 459.

4. Feizi, T., et al, TIBS (1985) 24 to 29.

5. Paulsen, H., et al, Carbohydr Res (1986) 146:147 to 153.

6. Sabesan, S., et al, J Amer Chem Soc (1986) 108:2068 to 2080.

7. Paulsen, H., et al, Carbohydr Res (1984) 125:47 to 64.

8. Paulsen, H., et al, Carbohydr Res (1985) 144:205 to 229.

9. Ogawa, T., et al, Eur Pat Appl Pub No 146090, Jun. 26, 1985.

10. Ogawa, T., et al, Tetrahedron Lett (1986) 27:5739 to 5742.

11. Pozsgay, V., et al, Carbohydr Chem (1987) 6:41 to 55.

12. Paulsen, H., et al, Carbohydr Res (1985) 137:63 to 77.

13. Ogawa, T., et al, Eur Pat Appl Pub No. 166442, Jan. 2, 1986.

14. Paulson, J. C., et al, Pure Appl Chem (1984) 56:797-806.

15. Loomes, L. M., et al, Nature (1984) 307:560-563.

Sialic acid glycosides are known to occur in a wide variety ofbiological materials¹,2 in the form of gangliosides and complexoligosaccharides attached to proteins. These are present in bodilyfluids and on cell surfaces. Sialic acid-containing structures have beenshown to be important for the attachment of viral particles to tissuesand protection of proteins from proteolysis. They are known to be higherin concentration³,4 in sera of cancer patients as opposed to normalindividuals; they also occur on the tissues of cancer patients.Specifically structures having as terminal tetrasaccharides 19-9 andsialo-X moieties are related to the cancerous state. Assays takingadvantage of this association are described in U.S. Pat. No. 4,471,057;and antibody production to tumors bearing these haptens in U.S. Pat. No.4,172,124.

In order to detect, quantify and study the tetrasaccharides (and theirprecursors and biosynthesis) it is advantageous to obtain them and theirantibodies in practical amounts. The availability of these moieties fromnature through isolation is tedious and results in limited quantities ofmaterial that must be purified for further use. Also, material that isobtained through isolation does not provide for useful modifiedstructures, such as synthetic antigens or immunoadsorbents.

An alternative to isolation of such interesting structures to providewell-defined materials for the study of biological actions is chemicalsynthesis. The chemical synthesis of sialosides in high anomeric purityand reasonable yields has remained a difficult challenge for chemists inthe recent past⁵,6.

There has been moderate success in chemically preparing sialosides with2-6 linkages, however most reaction conditions with various substratesgive anomeric mixtures (α/β equals near 1/1)⁷,8,9,10. A wide variety ofreaction conditions has been reported, including variation of substratealcohol, catalyst, and solvent. The result of these reactions is widevariation of overall yield of sialosides (10-80%) but generallyconsistent α/β ratios of near 1/1 with few exceptions⁷,11. Such mixturesare tedious and difficult to separate to obtain the desiredalpha-sialoside.

The reported methods for forming a glycosidic linkage between the twoposition of sialic acid and the three position of galactosides (2-3linkage) and derivatives of these, have been even less sucessful⁵.Overall yields of sialosides (α and β) are consistently lower andanomeric purity is poor. Again a wide variety of alcohols, catalysts andsolvents have been used in these attempts. As it has been shown⁷,12 thatthere is great variation obtained (both in anomeric specificity andoverall yield) with various acceptors, donors and reaction conditions inthe formation of 2-6 linkages, extrapolation from these results to theformation of 2-3 linkages in a meaningful way is difficult anduninstructive. The danger of such comparisons is well known to theskilled chemist.

All but five reported examples of sialoside synthesis of higheroligosaccharides use a step-wise synthetic strategy. The examples of"block synthesis" used to produce higher sialosides involving the use ofa 2-6 block show limited versatility, or poor anomericspecificity⁸,10,13.

The one reported example of the synthesis of a 2-3 block suffers fromthe same problems more severely. This block is produced through thereaction of a sialoside derivative with a 3,4 diol of a disaccharidewhich results in the 2-3 linkage in 17% yield with an α/β ratio of 0.4.This block has not been used for the synthesis of largeroligosaccharides to effect an intersugar linkage¹³.

The one consistent factor in all strategies for the synthesis of highersialosides is the use of a methyl ester as the temporary blocking groupfor the acid moiety of the sialosyl halide. Use of this group would seemsensible as it provides the necessary blocking while conferring minimalsteric interference adjacent to the carbon through which the glycosidiclinkage is to be formed, and it is believed that the inherent stericrestriction around carbon two of ketoses is, in part, also responsiblefor the increased production of undesirable unsaturated products duringglycosylation of sialic acid derivatives.

The use of a methyl ester derivative of the sialyl halogenose results inlimitation of subsequent use of the product oligosaccharide for theformation of synthetic antigens and immunoadsorbents which are among theobjects of this invention. This limitation is due to the desirability ofbeing able to easily deblock a synthetic sialoside, including its acidgroup, while maintaining an ester group present in a linking arm,attached to the oligosaccharide, for subsequent activation to allowcoupling of the sialoside to proteins and insoluble carriers. Suchcoupling is achieved for most oligosaccharides through attachment of asynthetic oligosaccharide to an amino or carboxylic acid group on aprotein carrier. The strategies of coupling to carboxylic acid groups inproteins are precluded, as it would be commonplace to use anamino-terminated linking arm. This would result in undesirableself-polymerization of the now carboxylic unprotected syntheticoligosaccharide. Therefore, terminally derivatized acid linking armswhich, by the nature of the ester or other derivative, can be chemicallydifferentiated from the ester used to block the sialyl acid group arepreferred, and these derivatives must be persistent through the totaldeblocking of the sialoside.

There are few reports in the prior art of the preparation of anysynthetic sialyl oligosaccharide antigens and immunoadsorbents or theproperties of these. There are no reports, to our knowledge, of higher(more than one or two different sugar residues) synthetic sialyloligosaccharide antigens or immunoadsorbents.

DISCLOSURE OF THE INVENTION

The invention provides efficient synthesis of sialosides in highanomeric purity to allow the preparation of synthetic antigens andimmunoadsorbents that are useful for the preparation, detection,purification or removal of antibodies, lectins, receptors or otherbiomolecules which have an affinity for such structures. The synthesisemploys a synthetic block which contains the critical glycoside linkageand which can be converted to a variety of end products. The blocks arethus synthetic sialosides that are readily converted to the products andtheir useful corresponding antigens and immunoadsorbents. A particularadvantage of the invention disclosed herein results from the use ofesters other than methyl for blocking the carboxyl of the intermediatesialohalogenose, such as the benzyl or phenacyl esters. Theseintermediates give a higher yield of product, and, specifically, yieldhigher desired anomeric purity in obtaining the α-sialoside blocks.

As the synthetic blocks disclosed herein are of a minimum size (sialosyldisaccharide); they can be used to synthesize any of the naturallyoccurring α (2-3) or α (2-6) sialosides, or alternative forms containingthese moieties. This approach also permits a wide range of linking arms,since the chemistry of the linking arm attached to the reducing end ofthe higher oligosaccharide does not significantly alter the attachmentof the block to additional sugars.

The synthetic method of the invention can be applied specifically to thesynthesis of certain sialyl-containing trisaccharides and to certaintetrasaccharides, especially to trisaccharide precursors and thetetrasaccharides designated 19-9 and sialo-X. The 19-9 and sialo-Xtetrasaccharides which exemplify the synthetic methods of the inventionare known to be associated with malignancy. Antibodies immunoreactivewith these haptens are useful as diagnostic and potential therapeutictools in the management of malignancy. The intermediate saccharides arealso substrates to assay the relevant glycosyltransferases.

All the synthetic haptens of the invention can be used in thepreparation of both antigens and immunosorbents. The immunosorbents areuseful for the purification and detection of antibodies reactive withthe haptens. The haptens, when attached to a solid support to formimmunosorbents, can be used to remove or purify these antibodies fromblood or plasma. In addition, the labeled haptens can also be used indiagnostic assays for substances containing the haptens or for theircomplementary antibodies.

Thus, in one aspect, the invention is directed to compounds containingmoieties of the formula: ##STR1## wherein Ac is acyl (C1-6),representing sialo-Lewis^(a) (19-9) and sialo-X, respectively. Thesemoieties may be prepared in isolated forms per se, and further may beconjugated to solid support, to an antigen-forming carrier, to a label,to another sugar residue, or to a linking arm useful in effecting suchconjugations. The intermediate trisaccharides are similarly useful; inaddition, the trisaccharides can serve as substrates for the assay ofsamples for the relevant glycosyltransferase. The invention includesthese trisaccharides and their derivatives.

In still another aspect, the invention is directed to a method to effecta high yield of the α anomeric 2-3 sialyl-galactopyranosyl linkage. Thismethod comprises reacting a sialyl halide wherein the acid function isblocked as an aromatic ester with a suitably protected galactopyranosylacceptor to give an additional synthetic block or useful end product.

In another aspect, the invention is directed to block intermediatesuseful in the synthesis of oligosaccharides containing an α (2-3) sialylgalactopyranoside unit. The invention is also directed to methods forproducing desired oligosaccharides using this intermediate block, and toadditional intermediates characteristic of this method. In still anotheraspect, the invention is directed to the formation of intermediate α(2-6) disaccharide blocks, and to the use of these blocks in furthersynthesis.

Thus, also included is a method to effect a high yield of the α anomeric(2-6) sialyl-galacto pyranosyl linkage, which method is analogous tothat leading to the α anomer of the (2-3) linked product. A furtheraspect is directed to the resulting block intermediate and to methodsfor producing desired oligosaccharides using this 2-6 intermediate blockand the associated intermediates involved in this further synthesis.

All of the foregoing intermediates may be characterized as "haptens" andare useful as described below.

In other aspects, the invention is directed to methods of using thetetra- and trisaccharide moieties or other haptens or their conjugatesfor diagnosis, isolation and purification procedures, and therapy.

In still other aspects, the invention is directed to methods ofpreparation of the haptens and of their conjugates, including the methodcomprising deprotecting the protected forms thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 43 illustrate the structure of compounds 1 through 43respectively.

FIG. 44 shows the formation of the α (2-3) sialyl block of theinvention.

FIG. 45 shows the synthetic scheme for the production of sialo-X.

FIG. 46 shows the synthetic scheme for the production of 19-9.

FIG. 47 shows the formation of the α (2-6) sialyl block of theinvention, and its use in synthesis.

MODES OF CARRYING OUT THE INVENTION

The invention provides as a convenient synthetic block, α (2-3) sialylgalactopyranoside, a disaccharide which occurs in a number of importantoligosaccharides. This block is useful in synthesis of a number ofoligosaccharides which are significant in various biological processes.The use of this intermediate block is illustrated in the preparation oftwo particular trisaccharides of the structures ##STR2## Thesetrisaccharides can then be converted to the tetrasaccharidessialo-Lewis^(a) (19-9) and sialo-X, the structures of which are shownabove.

All of the 19-9 and sialo-X and their trisaccharide intermediatescontain an α (2-3) sialyl-galactopyranosyl unit. This unit is linked β(1-3) or β (1-4) to N-acetyl glucosamine to give the trisaccharidicprecursors for the 19-9 and sialo-X tetrasaccharides, respectively.Further attachment of α-fucose (1-4) or (1-3) to these trisaccharidesprovide for the 19-9 and sialo-X, respectively.

The tri- and tetrasaccharides are produced in a manner which permitsthem to be easily converted into synthetic immunoadsorbents and antigensthat are useful for the preparation, isolation, detection andpurification of the corresponding antibodies. The (2-3) sialo-containingblock can also be used in moieties which can detect lectins orreceptors, such as bacterial and viral receptors, that serve asadhesins¹⁴,15.

The tetrasaccharide moieties illustrated are known to be associated withmalignant tissue. They can be prepared, as illustrated, alreadyconjugated to convenient linkers as shown in compounds 35 and 37 herein,to permit convenient attachment to chromatographic supports or toantigen forming carriers, or to labels.

In an analogous manner, a sialo disaccharide block having an α (2-6)glycosidic linkage is provided for subsequent conversions to highersaccharide moieties having utilities similar to those described abovefor the sialo α (2-3) block derived saccharides.

A. Definitions and Scope

As used herein, "protecting group" refers to moieties ordinarily used inoligosaccharide synthesis to prevent reaction of the hydroxyl groups inthe reactions being conducted. Suitable protecting groups include acylgroups, especially lower acyl such as propionyl and acetyl, aromaticacyl groups, such as benzoyl, silyl groups such as trialkyl- oralkylarylsilyl, in particular t-butyldiphenylsilyl, and acetalderivatives, all protecting for hydroxy moieties, and protecting groupsfor carboxylic moieties such as phenyl and benzyl. Any commonly usedprotecting group known in the art for use in oligosaccharide synthesisis included.

The saccharide haptens of the invention are shown in structurescontaining their essential features, and wherein the hemiacetal is ofthe form --OY rather than --OH. This convention is used because thesaccharide hapten may be derivatized in various ways. Thus, Y may besimply H or alkyl (C1-6). Y may also be an additional sugar orsaccharide, if the hapten is part of a larger carbohydrate. Y may be alinking arm useful to conjugate the hapten to solid supports or to othersubstances. Y may be or may include an immunogenicity-conferringsubstance, such as a protein or other "antigen-forming carrier" if thehapten is to be used as an antigen. Y may be or include achromatographic support if the hapten is to be used as animmunoadsorbent. Y may include a label if the compound is to be used inassays. Where Y includes a solid support, carrier or label, conjugationof the hapten to same can be through a linking arm, and embodimentswhich include a linking arm are preferred. The parameters describing Yin these various instances are set forth herein.

Convenient linking arms useful in conjugation of the haptens preparedaccording to the method of the invention are of the general formula:--X--CO--L, wherein X is a hydrocarbylene of 3-19C in which 1-3nonadjacent CH₂ may optionally be replaced by NR, S, or O (R is H oralkyl, C1-6), and wherein L is a leaving group, such as OR, NHNH₂, orN₃, or can be converted to a leaving group. The linking arm ispreattached to the relevant monosaccharide or disaccharide beforeincorporation into the oligosaccharide to be produced. For example, inthe illustrated tetrasaccharides of the invention, the acetylglucosamine monosaccharide (or its protected or derivatized precursor)is incorporated into the product with the linker conjugated to theoxygen in position 1. In analogous oligosaccharides prepared using theintermediate blocks of the invention, linking arm-conjugated mono- ordisaccharides are employed as appropriate.

Therefore, although the above embodiment of the linking arms of theinvention has been defined rather generally, alternative linkers couldalso be used, so long as they do not contain groups which are reactivewith the saccharide moieties or which interfere with the syntheticreactions to form the oligosaccharides. The use of the above derivatizedcarboxyl group as the functional group for attachment to the solidsupport or other desired conjugate is convenient, and a particularlypreferred leaving group is OR, i.e., the ester, particularly the methylester. The ester can, however, be converted to contain an alternativeleaving group such as --NHNH₂ or --N₃ after the oligosaccharide issynthesized.

A preferred embodiment for X is a straight chain alkylene of the formula(CH₂)_(n), wherein n is 3-19. Exemplified herein is the linking armwherein n is 8; however this is simply a choice of convenience. Thespacing between the antigenic moiety and the remainder of the conjugatemay be manipulated by adjusting the size of the linker. For use as animmunoadsorbent, the spacing generated by a linking arm of this formulawherein X=(CH₂)₆₋₉ may be particularly advantageous.

In two convenient applications, the hapten moiety may be conjugated to asolid support to serve as an immunoadsorbent or to an antigen-formingcarrier to serve as an immunogen. The linking arms are useful, but notalways entirely necessary in conjugating the hapten to the support orcarrier. It is preferred to employ a linking arm because suitablereactivity of the linker makes conjugation more convenient, but perhapsmore important, because the linking arm provides desired spacing betweenthe hapten and support or carrier.

A variety of solid supports may be used for the first purpose,especially aminated supports such as those derived from silica gel,various organosiloxane derivatives, derivatized polyacrylamide or otherresins, controlled pore glass, agarose and derivatized alumina. Othersolid supports consistent with the chemical nature of the antigen canalso be employed.

Suitable antigen forming carriers include proteins, such as theappropriate serum albumin, such as human or bovine serum albumin,keyhole limpet hemacyanin, tetanus toxoid, and the like. A variety ofcarriers which do not themselves raise interfering antibodies in theparticular host is known in the art.

If the saccharides of the invention are to be used in assays, they mayalso be supplied in labeled form.

Suitable labels include radioisotopes such as ³² P or ¹²⁵ I,fluorophores such as fluorescein or dansyl, chromophores and enzymes.Means to conjugate the hapten to label are known in the art, and mayinclude the use of the above linking arms.

B. Synthetic Methods

The tetrasaccharide and trisaccharide haptens of the inventioncontaining sialyl α (2-3) glycosides are obtainable in practicalquantities for the first time, due to the availability of a blockintermediate prepared by the method of the invention. The preparation ofthis block is shown in FIG. 44. It is an α (2-3) linkedsialylgalactopyranoside and the preferential preparation in the αanomeric form uses reaction of a suitably derivatized sialyl halide withgalactopyranosyl acceptor. The improvement in results over the methodsof the prior art resides at least in part in the use of a benzyl orphenacyl ester of the sialyl residue in place of the methyl ester usedpreviously. The presence of this derivatizing group preferentiallyresults in the α anomer.

The intermediate block compound is shown herein in various protectedforms as compounds 14, 15, 16, 18, 19, 20, 21 and 22. Compounds 14-16are the 1-acetyl derivatives of the galactopyranoside; compounds 18-21are the O-allyl or halide derivatives. Illustrative preparation of thesevarious forms of the intermediate block are set forth in Examples IV, V,and VI of the herein application.

As shown in FIG. 44, a suitably protected form of the O-allyl orO-acetyl galactopyranoside is reacted with an aromatic ester, e.g., thebenzyl ester of the polyacetylated (or otherwise protected)sialylchloride. The resulting block intermediate is prepared in the α anomericconfiguration in 47% total yield with only traces of the undesirable βanomer using the acetylated acceptor in Example IV and in 39% yieldusing the O-allyl galactopyranosyl receptor in Example V. In both cases,the benzyl ester of the sialyl halide is used as reagent.

The formation of the disaccharide block illustrated in FIG. 44, permitsits subsequent reaction to form a variety of saccharide moieties,including the tri- and tetrasaccharides of the invention.

FIGS. 45 and 46 illustrate the use of the intermediate block to obtaintrisaccharide precursors. The trisaccharide precursors are deblocked togive the trisaccharide product compounds. These are in turn converted.As shown in FIG. 45, a suitably protected form of the block is reactedwith the 3,4 diol of a glucosamine derivative which contains asilyl-derived protecting group in the 6-position. This reaction isconducted in a non-polar dry solvent in the presence of trimethylsilyltrifluoromethane sulfonate, added portion-wise over approximately onehour.

The reaction continued to completion over another approximately onehour. The use of this acceptor, having a t-butyldiphenylsilyl protectinggroup in the 6-position, surprisingly, favors the formation of thedesired β (1-4) linkage. This result is unexpected in view of the stericfactors. The resulting trisaccharide is then reacted with suitablyprotected fucopyranosyl bromide. This is freshly prepared from theprotected fucopyranose. The reaction occurs under conditions generallyknown in the art. The resulting compound having the backbone structureof sialo-X is then deprotected as required.

The procedure for preparation of a 19-9 as shown in FIG. 46, is similar,except that the acceptor used, which has a different protecting group atthe 6-position which, although sterically less significant, favors theformation of a trisaccharide with the β (1-3) linkage. Reactionconditions and catalysts are similar.

As shown in FIGS. 45 and 46, P represents a hydrogen or a protectinggroup as required and Y usually represents hydrogen, alkyl (1-6 C), alinking arm or another saccharide, but could also include a solidsupport, antigen-forming carrier or label. X₂ is used to indicate thatthe nitrogen of glucosamine can be in the form either of an azide or anacylated amino where acyl (Ac) is C1-6. Conversion of the azide to theprotected amino form is conducted at a convenient point in thesynthesis.

A block intermediate for the 2-6 linked sialyl galactopyranosides isprepared in a manner analogous to that shown in FIG. 44, except that thegalactopyranosyl derivative is protected in the 3, 4 and 5 positions andhas a free hydroxyl at position 6. This synthesis is shown in FIG. 47.Further conversion of this block intermediate to higher saccharides isanalogous to that described for the α (2-3) sialyl block.

C. Utility of the Sunthetic Haptens

The 19-9 and sialo-X haptens are known to be associated with malignanttissue. Accordingly, antibodies raised against these antigens are usefulin diagnosis and therapy. The antigens obtained from these haptens ofthe invention are useful in producing such antibodies, andimmunoadsorbents prepared from the haptens are useful in isolating,purifying, and detecting them. The trisaccharide intermediates can beused in analogous ways. In addition, they are useful as substrates forassessment of the activity of the relevant glycosyltransferases. Thelevel of activity of the transferases is of diagnostic significance aswell. For determination of transferase activity, the sample is contactedwith the invention trisaccharides in the presence of a suitablemonosaccharide acceptor and the decrease in concentration of thetrisaccharide(s) or increase in concentration of tetrasaccharide(s) isdetected.

In one application, the haptens of the invention are conjugated to anantigen-forming carrier and used to obtain large quantities of antiseraimmunoreactive with the antigens. Standard immunization protocols areused to prepare antisera in mammalian subjects such as mice, rabbits, orlarge animals such as sheep. Humans may also be used as subjects if itis intended that the antibodies be used in therapy, to preventinterspecies immune reactions. The antisera are harvested and useddirectly, or, if desired, the peripheral blood lymphocytes orspleenocytes of the immunized subject may be harvested forimmortalization. The resulting monoclonal-secreting cell lines formspecific antibodies of high affinity and specificity. An adequate supplyof hapten is required not only to obtain the immunogen, but also inorder to assay preparations for the production of the desired antibody.In such assays, the haptens or antigens may be used in labeled form.

In a further application, the haptens of the invention may be conjugatedto a solid support in order to isolate and purify antibodies from theantisera. In this method, the hapten moieties are conjugated to thechromatographic support solid, such as those set forth above, and thesample from which the antibodies are to be isolated is applied to thesupport which is preferably configured as a chromatographic column. Theantibodies are specifically adsorbed from the sample, and eluted by saltgradients, pH shifts or other methods known in the art.

The adsorption to and elution from chromatographic support results inisolation of or removal of antibodies from a sample, as desired, andalso in their purification.

In still another application, the haptens are useful as assay tools. Inthe most simple application, they can be used to assess the presence andamount of antibody in a sample such as a plasma or blood sample, or in asample extracted from tissue. This is an essential concomitant tool inthe preparation and assay of antisera and monoclonal antibodies specificfor the antigens, as well as a tool in diagnosis of relevant parameterssuch as enzyme levels (sialyl α (2-3) trisaccharides) or malignancy(tetrasaccharides).

A variety of immunoassay techniques are available and known in the artfor use in detecting antibodies. In a typical approach, the antigen isused to coat multi-well plates to which a sample suspected of containingthe antibody is applied. After the plates are washed, an additionallabeled antibody specific for the species to which the sample-containedantibodies belong is added to assess the retention of antibodies fromthe sample in the wells. Alternatively, the antigen, labeled asdescribed above, can be used to detect the antibodies directly, or canbe used in competitive immunoassays to measure antigen levels. In thislatter approach, the haptens or antigens of the invention are conjugatedto label and mixed with sample in competition with analyte containedtherein for the relevant antibodies.

A variety of other protocols and variations of assays which depend fortheir specificity on antigen/antibody binding are known in the art andare permitted by use of the haptens of the invention or the antibodiesgenerated therefrom.

With respect specifically to the trisaccharide compounds 27 and 30,which are the biological precursors of the 19-9 and sialyl-X structuresrespectively, these haptens and their antigens and the immunosorbentscan be used to study the nature of the reactivities of antibodies thathave affinities for the larger structures. They can also be used tostudy the reactivities of the fucosyl transferases that act on thenatural terminal structures to produce the terminal tetrasaccharidesthat confer the reactivities of the 19-9 and sialyl-X oligosaccharides.Assay systems that include the synthetic hapten coated on plastic whichis incubated with serum samples, cellular and tissue extracts or otherbiological preparations in the presence of fucosyl donors that arelabeled can be used to study the presence of such transferases. Thismethod can be used to quantify fucosyl transferases. The synthetictrisaccharidic haptens themselves can be used as soluble acceptors insimilar reactions and the product tetrasaccharides can be isolated andquantified by physical methods.

EXAMPLES

The examples below illustrate but do not limit the scope of theinvention.

Examples I and II demonstrate the synthesis of the derivatized sialicacid halides, compounds 3, 5, and 6, through the use of the benzyl andphenacyl esters and the conversion of the per-O-acetylated derivativesinto the halides.

Example III relates to the formation of α (2-6) linkage. In thisreaction, the α anomer is formed exclusively when the phenacyl ester ofthe sialosyl choride 5 is used whereas when the corresponding methylester is used, an α/β ratio of near 2/1 is obtained.

The improved anomeric control obtained when the benzyl or phenacyl esterof the sialosyl halide is used as a reagent is applied to the synthesisof the desired α (2-3) linked block in Example IV. Compound 14, aprotected form of the block, is obtained by reaction of the benzyl ester3 with the diol 13 of the derivatized galactose unit. Compound 14 isproduced in 47% yield with only traces of the undesirable β anomer or ofthe 2-2 sialoside. The formation of this structure is central to thetotal synthesis of the 19-9 and sialo-X structures, and of otheroligosaccharides containing the 2-3 linkage. Compound 14 is convertedinto compounds 15 or 16 which serve as the desired sialo (2-3)α-anomeric blocks. It can be used directly in subsequent glycosylationsto give the extended structures in high yield.

Example V shows an analogous synthesis of the intermediate wherein theacceptor structure is the allyl glycoside of galactose 17. Reaction withthe benzyl ester 3 leads to the activated block disaccharide 20. ExampleVI demonstrates the use of this block to obtain the trisaccharide 22which is a unit present in G_(Ms), G_(M1), and G_(D) structures.

Examples VII and VIII continue the synthesis directed to the desiredtetrasaccharides. These examples describe the synthesis of the lineartrisaccharides 27 and 30 and derivatized forms of these compounds thatare useful for the synthesis of the desired tetrasaccharides 35 and 37.Example VII details the preferred method for forming the (1-3) linkagebetween the block and a glucosamine derivative that is an intermediatein the synthesis of 19-9, compound 35. Example VIII shows the preferredmethod for the synthesis of the analogous trisaccharide having a (1-4)linkage that is a precursor for the production of the sialo-Xtetrasaccharide, compound 37.

Although both intermediate trisaccharides (the (1-3) and the (1-4)) areproduced using the common (2-3) block disaccharides with acceptorstructures 23 and 31, each of which is a 3,4-diol, the ratio of 1-3 and1-4 linkages formed is reversed depending on the choice of derivatizedacceptor. The acceptor 23, acetylated at position 6, favors (1-3); theacceptor 31, with the t-butyl (diphenyl) silyl group at position 6,favors (1-4), a surprising result in view of the steric factors.

Examples IV and X detail the synthesis of the desired tetrasaccharidesfrom the trisaccharide intermediates. The resulting 19-9 and sialo-X areconverted into synthetic antigens and immunoadsorbents as described inExamples XI and XII respectively.

Example XIII demonstrates the use of these antigens for the detection ofantibody. The use of the synthetic haptens and glycoconjugates asinhibitors in this example demonstrates the potential for using thisassay format for the detection of natural 19-9 structures through theinhibition of anti-19-9 reactive antibodies with the bound syntheticglycoconjugates.

EXAMPLE I Benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-β-D-glycero-D-galacto-2-nonulopyranosylonate(3)

Acetic anhydride (13.5 g, 13.2 mmol) and some dimethylaminopyridine wereadded to N-acetyl neuraminic acid (1) (5 g, 16.1 mmol) in pyridine (25ml). After stirring overnight at 22° C., TLC (chloroform:methanol:water65:35:8) indicated a complete reaction. After addition of some methanol(10 ml) the mixture was stirred for 1 hour and co-evaporated with anexcess of toluene. The residue was dissolved in ethyl acetate andtreated with some Dowex 50 resin (H⁺, 20 ml). The product which wasrecovered after filtration, evaporation and drying in vacuo, was useddirectly in the next step.

Potassium fluoride (2.3 g, 39.6 mmol) followed by benzyl bromide (4.00g, 23.4 mmol) were added to the above material (8.40 g) indimethylformamide (85 ml). After stirring overnight at 22° C. thesolvent was evaporated in vacuo, the residue taken in dichloromethane,filtered and washed with water (three times). The recovered crudeproduct was chromatographed on silica gel (250 g) using a mixture ofhexane, ethyl acetate and ethanol (6:4:1) giving the benzyl ester 2 asan α/β mixture (14:86, 8.25 g, 84%). The pure α and β anomers could beobtained through chromatography and were characterized as follows.

α anomer (foam): [α]²² _(D) +20.3°(c1.0, chloroform); ¹ H-nmr: 7.30(m,5H, aromatics), 5.293(dd, 1H, J₆,7 2.5 Hz, J₇,8 7.0 Hz, H-7), 5.138(m,3H incl. NH, PhCH and H-8), 5.038(d, 1H, J_(gem) 12.5 Hz, PhCH),4.893(ddd, 1H, J_(3e),4 4.6 Hz, J₄,5 10.4 Hz, J_(3a),4 11.6 Hz, H-4),4.670(dd, 1H, J₅,6 11.0 Hz, H-6), 4.288(dd, 1H, J₈,9a 2.7 Hz, J_(9a),9b12.5 Hz, H-9a). 4.088(m, 1H, H-5), 3.988(dd, 1H, J₈,9b 5.5 Hz, H-9b),2.485(dd, 1H, J_(3e),3a 14.0 Hz, H-3e), 2.060, 2.108, 1.990, 1.968,1.965, 1.818(6s, 19H, 5 OAc, 1 NAc, H-3a).

Anal. Calc. for C₂₈ H₃₅ O₁₃ N: C, 56.65; H, 5.94; N, 2.36. Found: C,56.52; H, 5.88; N, 2.15.

β anomer: m.p. 128° C.; [α]²² _(D) -29.5°(c1.0 chloroform); ¹ H-nmr:7.35(m, 5H, aromatics), 5.375(dd, 1H, J₆,7 2.0 Hz, J₇,8 5.6 Hz, H-7),5.263(m, 3H incl. NH, H-8 and PhCH), 5.168(d, 1H, J_(gem) 12.0 Hz,PhCH), 5.093(ddd, 1H, J₈,9a 2.6 Hz, J₈,9b 6.5 Hz, H-8), 4.450(dd, 1H,J_(9a),9b 13.0 Hz, H-9a), 4.138(m, 3H incl. H-5, H-6 and H-9b),2.253(dd, 1H, J_(3e),4 5.0 Hz, J_(3e),3a 13.5 Hz, H-3e), 2.125,2.113(two), 2.025(two), 1.895(19H, 5 OAc, 1 NAc, H-3a).

Anal. Calc. for C₂₈ H₃₅ O₁₃ N: C, 56.65; H, 5.94; N, 2.36. Found: C,56.58; H, 5.82, N, 2.18.

Concentrated hydrochloric acid (1.3 ml) was added dropwise to the cooled(-20° C.) solution of compound 2 (5.28 g, 8.66 mmol) and acetyl chloride(11.73 g, 10.7 ml, 14.9 mmol) in dichloromethane (25 ml). The mixturewas stirred at 22° C. overnight in the tightly closed flask. TLC(chloroform, acetone 3:2) indicated a complete reaction. After cooling,the flask was opened and the content diluted with cold dichloromethane(100 ml) and quickly washed with cold water (20 ml), a cold solution ofsodium bicarbonate (20 ml), cold water (20 ml) and brine (20 ml). Dryingover magnesium sulfate at 5° C. and evaporation of the solvent left thecrude material 3 (5.07 g, 98%, foam) which showed very little impurity(¹ H-nmr) and was used directly as described in Example IV. ¹ H-nmr:7.40(m, 5H, aromatics), 5.395(dd, 1H, J₆,7 2.4 Hz, J₇,8 6.4 Hz, H-7),5.322(ddd, 1H, J_(3e),4 4.8 Hz, J_(3a),4 =J₄,5 10.9 Hz, H-4), 5.305(d,1H, J_(gem) 12.5 Hz, PhCH), 5.155(d, 1H, PhCH), 5.105(ddd, 1H, J₈,9a 2.6Hz, J₈,9b 6.2 HZ, H-8), 4.343(dd, 1H, J_(9a),9b 12.4 Hz, H-9a),4.293(dd, 1H, J₅,6 9.7 Hz, H-6), 4.135(m, 1H, H-5), 4.012(dd, 1H, H-9b),2.770(dd, 1H, J_(3a),3e 14.5 Hz, H-3e), 2.282(dd, 1H, H-3a), 2.115,2.040(two), 2.013, 1.902(15H, 4 OAc, 1 NAc).

EXAMPLE II Phenacyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-trideoxy-D-glycero-.beta.-D-galacto-2-nonulopyranosylonate(5)

N-Acetylneuraminic acid (1) (2.0 g, 6.47 mmol) was treated at ambienttemperature with pyridine (9 mL) and acetic anhydride (3 mL) with acatalytic amount of dimethylaminopyridine for 16 h. The originallycloudy suspension goes clear in time. TLC developed with ethylacetate:methanol 4:1 shows the reaction is complete. The solvents wereremoved by evaporation under high vacuum to yield a light brown residuewhich was dissolved in ethyl acetate (.sup.˜ 20 mL) and IR-120(H+) resin(5 mL) added and stirred for 5 minutes. Filtration and evaporation ofthe filtrate gave the crude product (3.360 g).

The crude per-O-acetylated N-acetylneuraminic acid (2.89 g, 5.49 mmol)was dissolved in anhydrous dimethylformamide (50 mL) to which was addedanhydrous potassium fluoride (0.710 g, 12.27 mmol), and recrystallizedphenacyl bromide (1.70 g, 8.54 mmol). TLC examination with hexane:ethylacetate: ethanol 6:4:1 development on silica gel showed the reaction tobe complete in 0.5 to 1.0 hours. The reaction mixture evaporated to givea residue (4.74 g) was taken up in dichloromethane, filtered and thefiltrate evaporated.

Column chromatography on silica gel (75 g) eluted with hexane:ethylacetate:ethanol 6:4:1 gave compound 4 (2.437 g). This represents a 62%yield based on the amount of N-acetylneuraminic acid used. Otherproducts that are produced and separated from the per-O-acetylatedphenacyl ester appear to be a very small amount of the α-anomericacetate and a small amount of 2,3 unsaturated compound.

¹ H-nmr: 7.90, 7.55(2 m,5H, aromatics), 5.56(s, 2H, --OCH₂ CO), 4.60(dd,1H, J 2,3 Hz, J 13.0 Hz, H-9), 2.66(dd, 1H, J_(3a),3e 14.0 Hz, J_(3e),45.6 Hz, H-3e), 2.37(t, 1H, J 11.0 Hz, H-3a).

Compound 4 (0.60 g) was treated with glacial acetic acid saturated withhydrogen chloride (25 mL, 4° C.) for 3.5h. TLC examination of thereaction mixture developed with ethyl acetate: hexane 3:1 showed thereaction to be complete. The mixture was diluted with toluene (20 mL)and evaporated to dryness under vacuum. The residue so obtained wascolumn chromatographed on silica gel (15 g) eluted with above solventmixture to yield compound 5 (0.40 g) in 69% isolated yield, ¹ H-nmr:7.92, 7.64, 7.52(3 m, 2H, 1H, 2H, aromatics) 5.59(d, 1H, J_(gem) 16.0Hz, --OCH₂ --CO) 4.42(d, 1H, --OCH₂ CO), 4.36(bd, 1H, J_(NH),5 10.0 Hz,NHAc), 4.21(m, 1H, H-4), 2.87(dd, 1H, J_(3e),4 5.0 Hz, H3e), 2.46(dd,1H, J_(3e),3a 14 Hz, J_(3a),4 11.5 Hz, H-3a), 2.16, 2.09, 2.06, 2.05,2.05(5s, 5Ac).

Phenacyl5-acetamido-4,7,8,9-tetra-O-acetyl-2-bromo-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosylonate(6)

The purified per-O-acetylated phenacyl ester of sialic acid 4 (0.051 g,0.079 mmol) was treated at ambient temperature with commercial glacialacetic acid saturated with hydrogen bromide, for a period of 0.5 h. Atthat time the solution was diluted with toluene and evaporated undervacuum. This crude product was column chromatographed on silica gel (7.0g), eluted with ethyl acetate:hexane 3:1 to afford the pure compound 6(0.025 g) in 47% yield as a clear syrup: ¹ H-nmr: 7.92, 7.64, 7.52,(3 m,5H, aromatics), 5.62(d, 1H, J_(gem) 15.5 Hz, --OCH₂ CO--), 5.52(dd, 1H,J₇,8 7.0 Hz, H-7), 5.42(d, 1H, --OCH₂ CO--), 5.38(d, 1H, J_(NH),5 9.2Hz, NHAC), 5.19(m, 1H, H-8), 4.45(dd, 1H, J₈,9a 2.9 Hz, J_(9b),9a 9.6Hz, H-9a), 4.33(dd, 1H, J₆,7 2.2 Hz, H-6), 4.29(dd, H, J 10.2 Hz, H-5,4.06(dd, 1H, J_(8b),9 5.5 Hz, H-9b), 3.00(dd, 1H, J_(3e),4 4.5 Hz,J_(3e),3a 14.0 Hz, H-3e), 2.44(dd, 1H, J_(3a),4 11.0 Hz, H-3a), 2.15,2.09, 2.06, 2.04, 1.93,(5s, 15H, 5Ac).

EXAMPLE III 8-Methoxycarbonyloctyl (methyl5-acetamido-3,5-dideoxy-D-α-glycero-D-galacto-2-nonulopyranosylonate)-(2-6)-β-D-galactopyranoside(9)

A mixture of8-methoxycarbonyloctyl-2,3-di-O-benzoyl-β-D-galactopyranoside (7) (0.056g, 0.101 mmol), silver trifluoromethanesulfonate (0.003 g, 0.011 mmol),silver carbonate (0.068 g, 0.248 mmol) 4° A molecular sieves (0.200 g,powdered), and dry dichloromethane (2 mL) was cooled to -20° C. under anitrogen atmosphere. The chloride 5 (0.060 g, 0.100 mmol) dissolved indichloromethane was added to the above mixture. The resulting mixturewas allowed to warm to -10° C. and held there for 1 h at which time TLCdeveloped with ethyl acetate:hexane 4:1 showed no apparent consumptionof halide or alcohol. The mixture was allowed to warm to ambienttemperature over a period of 4 hours. The reaction was observed toproceed slowly at ambient temperature and over a 15 h period all of thechloride 5 and a large portion of the alcohol 7 were consumed. A newmajor compound was produced which travelled slightly slower then thechloride when the plate was developed with hexane:ethyl acetate:ethanol10:10:1. At that time the mixture was filtered and the filtrateevaporated to dryness to give a white foam (0.111 g). This material wascolumn chromatographed (7 mm I.D., 35 mm) on silica gel (˜10 g) (200psi, 1.5 min fractions) eluted with hexane:ethyl acetate:ethanol10:10:1. The major fraction (f11-14) provided the product (0.086 g).Analysis of a 200 MHz ¹ Hmr spectrum of this material showed it to be asingle glycoside of purity greater the .sup.˜ 95%. The only contaminantobservable was the 2,3-ene compound resulting from elimination of the3-deoxy-2-chloro compound. The yield of this single glycoside, compound8 based on alcohol used was 70-72%: ¹ H-nmr: 5.78(dd, 1H, J₁,2 8.0 Hz,H-2), 5.50(dd, 2H, J_(gem) 14 Hz, OCH₂ CO--), 4.67(d, 1H, H-1), 3.12(d,1H, J₄,OH 5.5 Hz, OH), 2.78(dd, 1H, J_(3'e),4' 4.7 Hz, J_(3'e),3'a 12.5Hz, H-3'e), 2.18, 2.04, 1.98, 1.93, 1.91(5 s, 15H, 5 Ac).

The proof that this blocked phenacyl ester glycoside was the desiredα-anomer was directly obtained by treating a portion of the product (5mg) with sodium methoxide in methanol. This effected removal of theester blocking groups and transesterification of the phenacyl ester toprovide the methyl ester. De-ionization and removal of the resin byfiltration and evaporation of the filtrate gave a residue. Analysis of a200 MHz ¹ Hmr spectrum of this material showed it to be identical tothat of 8-methoxycarbonyloctyl (methyl5-acetamido-3,5-dideoxy-D-α-glycero-D-galacto-2-nonulopyranosonate)-(2-6)-β-D-galactopyranoside(9). As both anomeric (2-6) methyl ester glycosides have been preparedand their ¹ Hmr spectra fully assigned in the literature compound 8,when deblocked and transesterified, must be the α-anomer. The spectrumof the transesterified deblocked product showed no evidence of thepresence of the β-anomer. The ¹ Hmr (D₂ O) of the product contained thefollowing easily assignable peaks: 4.38(d, 1H, J₁,2 7.5 Hz, H-1),3.88(s, 3H, CH₃), 3.69(s, 3H, CH₃), 2.74(dd, 1H, J_(3'e),3'a 12.5 Hz,J_(3'e),4' 4.5 Hz, H-3'e), 2.04(s, 3H, NHAc), 1.86(t, 1H, J_(3'a),4'12.5 Hz, H-3'a).

EXAMPLE IV PREPARATION OF BLOCK UNITS, COMPOUNDS 15 AND 161-O-acetyl-4,6-O-paramethoxybenzylidene-β-D-galactopyranose (13).

Tetra-O-benzyl galactopyranose 10 (4.00 g, 7.41 mmol),dicyclohexycarbodiimide (4.90, 24.01 mmol) and a small amount of cuprouschloride were heated at 85° C. for one hour. After cooling in ice,acetic acid (1.4 ml) in dimethoxyethane was added dropwise to themixture which was stirred for one hour at 22° C. A solution of oxalicacid dihydrate (3.20 g, 25.0 mmol) in acetone (20 ml) was then added.Most of the acetone was evaporated off and the residue taken in etherand filtered. The organic solvant was washed with water, dried andevaporated leaving a residue which was crystallized from 98% ethanolgiving 11 (2.34 g, 56%): m.p. 101°-102° C.; [α]²⁰ _(D) +5.3°(c1.0,chloroform); ¹ H-nmr: 5.50(d, 1H, J₁,2 8.5 Hz, H-1), 1.98(s, 3H, O Ac).

Anal. Calc. for C₃₆ H₃₈ O₇ : C, 74.29; H, 6.57. Found: C, 73.55: H,6.37.

A suspension of 11 (2.20 g, 3.78 mmol) in acetic acid (35 ml) washydrogenated for 3 hours at atmospheric pressure in the presence of 5%palladium on carbon. The catalyst was separated and further washed withacetic acid. Freeze drying left a crude product which was crystallizedfrom 98% ethanol giving 12 (0.756 g, 92%): m.p. 173°-174° C.; ¹ H-nmr(D₂O): 5.61(d, 1H, J₁,2 8.0 Hz, H-1), 4.072(dd, 1H, J₄,5 1.0 Hz, J₃,4 3.0Hz, H-4), 2.20(s, 3H, O Ac).

Anal. Calc. for C₈ H₁₄ O₇ : C, 43.24; H, 6.35. Found: C, 42.93; H, 6.33.

Paratoluene sulfonic acid (0.020 g) was added to a suspension of 12(0.370 g, 1.67 mmol) and paramethoxy dimethoxytoluene (0.344 g, 1.90mmol) in acetonitrile (12 ml). After 0.5 hour, some triethylamine wasadded and the solvent evaporated in vacuo. After chromotagraphy onsilica gel using a mixture of chlorform and acetonitrile (4:6), 13 wasrecovered as a solid (0.475 g, 82%): ¹ H-nmr(D₆ DMSO): 7.38 and 6.43(2M,4H, aromatics), 5.50(s, 1H, benzylidene), 5.41(d, 1H, J₁,2 7.5 Hz, H-1),3.76(s, 3H, O--CH₃), 2.10(s, 3H, O Ac).

(Benzyl) 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxyα-D-glycero-D-galacto-2-nomulopyranosylonate)-(2-3)-O-(1-O-acetyl-4,6-O-paramethoxybenzylidene-β-D-galactopyranose)(14)

A solution of silver trifluoromethanesulfonate (0.924 g, 3.60 mmol) and2,6-di-tert-butylpyridine (0.768 g, 0.900 ml, 4.02 mmol) intetrahydrofuran (4 ml, distilled before being used on to molecularsieves) was added to a suspension of the diol 13 (1.188 g, 3.49 mmol) intetrahydrofuran (4 ml). Dissolution occurred after stirring. calciumsulfate (1.50 g crushed) was added and stirring was continued for 0.5hour at 22° C. After cooling at -78° C., the chloride 3 (2.25 g, 3.846mmol) in tetrahydrofuran (4 ml) was syringed in slowly (1 hour). TLC(chloroform:acetone 70:30 and hexane:ethyl acetate:ethanol 15:10:1, fourelutions) indicated a slow reaction. The mixture was stirred for 1 hourat -78° C., warmed up and stirred for 1 hour at -55° C. After coolingdown to -78° C., more silver triflate (0.514 g, 2.00 mmol) and2,6-di-tert-butylpyridine (0.382 g, 0.448 ml, 2.00 mmol) intetrahydrofuran (2 ml) were added followed by a slow addition of thechloride (1.17 g, 2.00 mmol) in tetrahydrofuran (1.5 ml). After furtherstirring for 1 hour at -55° C., the mixture was slowly warmed up to 0°C. in about three hours. After dilution with dichloromethane (100 ml)and filtration, the solvents were washed with a solution of sodiumbicarbonate, water and brine. The recovered crude product (5.74 g) waschromatographed on silica gel (240 g, 3.5×50 cm) eluted with a mixtureof chloroform and acetone (75:25, containing 0.01% of pyridine). Purityof the fractions was checked by running the TLC plates in both solventmixtures indicated above. This afforded the α-sialoside 14 (foam, 1.48g, 47%): [α]²² _(D) +7.6°(c1.0 chloroform); ¹ H-nmr: 7.400(m, 7H,aromatics), 6.970(m, 2H, aromatics), 5.625(d, 1H, J₁,2 8.0 Hz, H-1),5.540(ddd, J_(7'),8' 8.5 Hz, J_(8'),9'a 2.5 Hz, J_(8'),9'b 6.5 Hz,H-8'), 5.300[m, 2H incl. PhCH(d, J_(gem) 12.0 Hz) and H-7'(dd, J_(6'),7'2.5 Hz)], 5.175(d, 1H, J_(5'),NH 9.5 Hz, NH), 5.075(d, 1H, PhCH),4.975(ddd, 1H, J_(3'e),4' 4.5 Hz, J_(3'a),4' 12.5 Hz, J_(4'),5' 10.0 Hz,H-4'), 4.880(s, 1H, benzylidene), 3.900(ddd, 1H, J₂,OH 1.5 Hz, J₂,3 10.0Hz, H-2), 3.787(s, 3H, OCH₃), 2.785(dd, 1H, J_(3'a),3'e 14.0 Hz, H-3'e),2.192, 2.150, 2.130, 2.050, 2.017, 1.895(6 s, 19 H, 5 OAc, 1 NAc andH-3'a).

Anal. Calc. for C₄₂ H₅₁ NO₂₀ : C, 56.69; H, 5.88; N, 1.60. Found: C,56.23; H, 5.88; N, 1.60.

(Benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(1,2,4,6-tetra-O-acetyl-β-D-galactopyranose)(15)

Compound 14 (1.80 g, 2.02 mmol) was warmed up to 45° C. in a 10:1mixture of acetic acid and water (60 ml). TLC (ethyl acetate:methanol100:5) indicated the completion of the reaction in about 1.5 hours. Thesolution was co-evaporated with an excess of toluene and the residuedried in vacuo. Acetic anhydride (2 ml) and some DMAP were added to theabove material dissolved in pyridine (25 ml). After stirring for 18hours at 22° C., TLC indicated the completion of the reaction.

The residue obtained after evaporation of the solution with an excess oftoluene was diluted with dichloromethane. The solution was washed withwater, a solution of sodium bicarbonate, water and brine. The recoveredsyrup was combined with a material previously obtained in the samemanner from 14 (1.028 g, 1.156 mmol). This crude product (3.5 g) waschromatographed on silica gel (100 g) using ethyl acetate as eluant.Pure 15 (2.62 g, 92%) was obtained as a foam, [α]²² _(D) +27.2°(c1.0chloroform); ¹ H-nmr: 7.40(m, 5H, aromatics), 5.825(d, 1H, J₁,2 8.0 Hz,H-1), 5.513(ddd, 1H, J_(7'),8' 8.3 Hz, J_(8'),9'a 2.5 Hz, J_(8'),9'b 6.5Hz, H-8'), 5.488(d, 1H, J_(gem) 12.0 Hz, PhCH), 5.313(dd, 1H, J_(6'),7'2.5 Hz, H-7'), 5.165(dd, 1H, J₂,3 10.0 Hz, H-2), 5.087[m, 2H incl.PhCH(J_(gem) 12.0 Hz) and H-4(bd, J₃,4 3.5 Hz)], 4.938(d, 1H J_(5'),NH10.0 HZ, NH), 4.862(ddd, 1H, J_(3'e),4' 4.5 Hz, J_(3'a),4' 12.5 Hz,J_(4'),5' 10.0 Hz, H-4'), 4.787(dd, 1H, H-3), 3.533(dd, 1H, J_(5'),6'10.5 Hz, H-6'), 2.638(dd, 1H, J_(3'a),3'e 13.5 Hz, H-3'e), 2.213, 2.188,2.113, 2.088, 2.063(two), 2.043, 2.040, 1.982(8 s, 27H, 8 OAc, 1 NAc),1.687(t, 1H, H-3'a).

Anal. Calc. for C₄₀ H₅₁ O₂₂ N: C, 52.92; H, 5.66; N, 1.50. Found: C,53.27; H, 5.92; N, 1.47.

Preparation of (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(1,4,6,-tri-O-acetyl-2-O-benzoyl-β-D-galactopyranose)(16)

Benzoic anhydride (0.419 g, 1.85 mmol) and a small amount ofdimethylaminopyridine were added to the starting material 14 (0.713 g,0.801 mmol) in pyridine (5 ml). The mixture was stirred at 40° C. for 24hours and 2 hours at 22° C. after addition of some methanol. Dilutionwith dichloromethane, washing with water, a solution of sodiumbicarbonate and water followed by drying and evaporation of the solventsleft a residue which was co-evaporated with some toluene. The crudeproduct was chromatographed on silica gel (20 g) using a mixture ofchloroform and acetone (85:15) as eluant. The pure 2-O-benzoyldisaccharide (0.710 g, 89%) was obtained. ¹ H-nmr: 8.08(m,2H),7.20-7.60(m,5H) and 6.85(m,2H) all aromatics, 5.99(d, 1H, J₁,2 8.0 Hz,H-1), 5.56[m, 2H, incl. H-2(dd, J₁,2 8.0 Hz, J₂,3 10.0 Hz) and H-8'(m)],5.22(dd, 1H, J_(7'),8' 8.0 Hz, J_(6'),7' 2.0 Hz, H-7'), 5.08(d, 1H,J_(gem) 12.0 Hz, PhCH), 5.00[m,3H, incl. benzylidene(s), NH(d),PhCH(d)], 4.85(m, 1H, H-4'), 3.79(s, OCH₃), 2.69(dd, 1H, J_(3'a),3'e13.0 Hz, J_(3'e),4' 4.5 Hz, H-3'e), 2.23, 2.09, 1.99, 1.97, 1.82, 1.72(6s, 19H, 5 OAc, 1 NAc overlapping with H-3'a).

The above disaccharide (0.350 g, 0.357 mmol) was dissolved in a mixtureof acetic acid and water (90:10, 10 ml) and warmed up to 45° C. for 1hour. The mixture was co-evaporated with an excess of toluene and theresidue dried in vacuo (0.695 g).

Acetic anhydride (0.300 m) and some dimethylaminopyridine were added tothe above material dissolved in pyridine (4 ml). After 24 hours at 22°C., some methanol was added and the mixture worked up as usual leaving acrude material which was co-evaporated with toluene. The residue waschromatographed on silica gel using a mixture of chloroform and acetone(85:15) as eluant giving 16 (0.595 g, 88%). ¹ H-nmr: 6.05(d, 1H, J₁,28.0 Hz, H-1), 5.54(m, 1H, H-8'), 5.47(d, 1H, J_(gem) 12.0 Hz, PhCH),5.39(dd, 1H, J₂,3 10.0 Hz, H-2), 5.17[m, 2H, incl. H-4 and H-7'),5.08(d, 1H, PhCH), 4.92(dd, 1H, J₃,4 3.5 Hz, J₄,5 10.5 Hz, H-3), 4.80(m,2H incl. NH and H-4'), 2.59(dd, 1H, J_(3'e),4', 4.0 Hz, J_(3'e),3'a 13.0Hz, H-3'e), 2.21, 2.16, 2.12(two), 2.00, 1.98, 1.79, 1.45(8 s, 24H, 7OAc, 1 NAc), 1.73(t, 1H, J_(3'a),4' 12.0, H-3'a).

EXAMPLE V Preparation of allyl (benzyl 5-acetamido-4,7,8,9,-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-(4,6-O-benzylidene-β-D-galactopyranoside)(18)

The diol 17 (0.724 g, 2.35 mmol) obtained by well known procedures fromallyl β-D-galactopyranoside, silver trifluoromethanesulfonate (0.637 g,2.48 mmol), 2,6-di-tert-butlypyridine (0.523 g, 2.74 mmol) and calciumsulfate (0.500 g) were mixed in tetrahydrofuran (3 mL) as indicatedpreviously for the preparation of 14 in Example IV. After cooling at-45° C., the chloride 3 11.52 g, 2.61 mmol) in tetrahydrofuran (3 mL)was syringed dropwise in about 2 hours. The mixture was stirred for 1hour at -35° C. After cooling down to -45° C., more silvertrifluoromethanesulfonate (0.318 g, 1.24 mmol), and base (0.261 g, 1.37mmol) in tetrahydrofuran (1.5 mL) were added, followed by the chloride 3(0.800 g, 1.35 mmol) in tetrahydrofuran (2.5 mL) as indicated above.After stirring for 1 hour at -35° C., the mixture was slowly warmed upto 0° C. A work up similar to that described above for 14 provided acrude mixture (3.9 g). Recovery of the appropriate fractions obtainedafter chromatography on silica gel (100 g) using a mixture ofchloroform, acetone, and methanol (90:10:1) gave a mixture of thedisaccharide 18 and of the product of hydrolysis of the chloride.Further chromatography of silica gel 60H using a mixture of haexane:ethyl acetate: ethanol 15:10:1 (80 g) provided 18 (0.783 g, 39%). ¹H-nmr 6.00(m, 1H, --CH═), 5.49(m, 1H, H-8'), 5.18[m, 2H, incl. PhCH(d,J_(gem) 12.0 Hz)], 5.07(d, 1H, Ph CH), 4.98(s, 1H, benzylidene),4.95(ddd, 1H, J_(3'e),4' 4.5 Hz, J_(3'a),4' 13.0 Hz, J_(4'),5' 10.0 Hz,H-4'), 2.81(dd, 1H, J_(3'a),3'e 4.5 Hz, H-3'e), 2.23, 2.17, 2.06, 2.03,1.92(5s, 16H, 4 OAc, 1 NAc overlapping with H-3'e).

Preparation of Allyl (benzyl5-acetamido-4,7,8,9-tectra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-(4,6-di-O-acetyl-2-O-benzoyl-β-D-galactopyranoside)(19)

Benzoylation of 18 (0.600 g, 0.7 mmol) as described above in the case of16 gave a crude product. Chromatography on silica gel (18 g) using amixture of chloroform and methanol (97.5:2.5) as eluant gave the pure2-benzoyl disaccharide (0.630 g, 93%). ¹ Hmr: 5.78(m, 1H, --CH═),5.44(m, 2H, incl. H-8' and H-2(dd, J₁,2 8.0 Hz, J₂,3 10.0 Hz, H-2),4.78[m, 2H, incl. H-1 (d)], 2.78(dd, 1H J_(3'e),4' 4.5 Hz, J_(3'e),3'a13.0 Hz, H-3'e), 2.27, 2.08, 1.95, 1.83, 1.73(5 s, 16H, 4 OAc, 1 NAcoverlapping with H-3'a).

The above disaccharide (0.600 g, 0.624 mmol) was hydrolyzed (95° C.) andperacetylated as indicated previously for 16. The residue obtained waschromatographed on silica gel (18 g) using a mixture of chloroform andmethanol (98:2) as eluant giving 19 (0.502 g, 83%). ¹ H-nmr: 5.82(m, 1H,--CH═), 5.60(m, 1H, H-8'), 5.48(d, 1H, J_(gem) 12.0 Hz, PhCH), 5.33(dd,1H, J₁,2 8.0 Hz, J₂,3 10.0 Hz, H-2), 2.57(dd, 1H, J_(3'e),3'a 13.5 Hz,J_(3'e),4' 4.5 Hz, H-3'e), 2.23, 2.13(two), 2.08, 1.95, 1.76, 1.45, (6s, 21H, 6 OAc, 1 NAc), 1.70(t, 1H, J_(3'e),4' 13.0 Hz, H-3'e).

Preparation of (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(4,6-di-O-acetyl-2-O-benzoyl-β-D-galactopyranosylchloride) (20)

Palladium dichloride (0.046 g, 0.261 mmol) was added to a mixture of thestarting material 19 (0.250 g, 0.261 mmol) with sodium acetate (0.060 g,0.726 mmol) in a mixture of acetic acid (2.5 mL) and water (0.125 mL).After 20 hours at 22° C., the mixture was diluted with methylenechloride and the catalyst separated. The solvent was washed with water,a solution of sodium bicarbonate and water. Drying and evaporation lefta residue (0.240 g) which was chromatographed on silica gel (16 g) usinga mixture of chloroform and methanol (97:3), to give the product (0.163g, 67%) as an anomeric mixture (¹ H-nmr).

A solution of oxalyl chloride (0.011 g, 0.0087 mmol in methylenechloride was added to a solution of the reducing disaccharide (0.080 g,0.0871 mmol), dimethylformamide (0.0635 g, 0.871 mmol) in methylenechloride (2 mL) at -15° C. The mixture was slowly warmed up to -5° C.and a second portion of the chloride was added. After 0.5 hour, thetemperature was brought to 0° C. and a third portion of oxalyl chloridewas added. After 15 minutes at 0° C., the mixture was co-evaporated withan excess of dry toluene and dried in vacuo to give 20 (0.080 g). Asindicated by ¹ H-nmr the product contained about 15-20% of impurities. ¹H-nmr: 5.65[m, 2H, incl. H-1(d, J₁,2 8.0 Hz) and H-8'(m)], 5.49[m, 2H,incl. H-2(dd, J₂,3 10.0 Hz)], 5.44 (d, 1H, J_(gem) 12.0 Hz, PhCH),4.95(ddd, J_(3'e),4' 4.5 Hz, J_(3'a),4' 13.0 Hz, J_(4'),5' 10.0 Hz,H-4'), 2.70(dd, 1H, J_(3'e),3'a 12.5 Hz, H-3'e), 2.26, 2.23, 2.15, 2.14,2.10, 1.97, 1.40, (7s, 6 OAc, 1 NAc), 1.65(t, 1H, J_(3'a),3'e 12.5 Hz,H-3'a).

EXAMPLE VI Preparation of allyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(2,6-di-O-acetyl-β-D-galactopyranoside)(21)

Compound 18 (0.150 g, 0.175 mmol) in pyridine (1 ml) was acetylated inthe presence of dimethylaminopyridine for 24 hours at 22° C. Afteraddition of methanol, the usual work up left a residue which waschromatographed on silica gel (8 g) using a mixture of chloroform andacetone (70:30) as eluant giving the product (0.150 g, 95%). ¹ H-nmr:5.90(m, 1H, --CH═, 5.57(m, 1H, H-8'), 5.37(dd, 1H, J_(6'),7' 2.5 Hz,J_(7'),8' 9.5 Hz, H-7'), 5.24[m, 2H incl. H-2(dd, J₁,2 8.0 Hz, J₂,3 9.5Hz)], 4.90(s, 1H, benzylidene), 4.83(m, 1H, H-4'), 4.62(d, 1H, J₁,2 8.0,H-1), 2.75(dd, 1H, J_(3'e3'a') 4.5 Hz, J_(3'e),4' 12.5 Hz, H-3'e), 2.25,2.22, 2.12, 2.07, 2.03, 1.88,(6s, 5 OAc, 1 NAc, overlapping with H-3'a).

The above product was heated at 95° C. for 1 hour in a mixture of aceticacid and water (9:1, 4 ml). Co-evaporation with toluene and drying invacuo left a residue which was run through silica gel (3 g) using amixture of chloroform and acetone (65:35) giving 0.098 g (72.5%) of the4,6-diol.

Acetyl chloride (5.6 mg, 0.071 mmol) in methylene chloride (0.100 ml)was added to a mixture of the diol (0.057 g, 0.071 mmol), pyridine(0.0062 mg, 0.078 mmol) in dichloromethane (5 ml) and cooled to -70° C.The mixture was slowly warmed up to 0° C. for 1 hour. After cooling downto -78° C. more pyridine (0.012 g, 0.156 mmol) followed by acetylchloride (11.2 mg, 0.142 mmol) in dichloromethane (0.200 ml) were added.The mixture was then warmed up to -15° C. and methanol added. After theusual work up followed by drying and evaporation of the solvents, theresidue was chromatographed on silica gel (2.5 g) using a mixture ofchloroform and acetone (75:25). Evaporation of the appropriate fractionsprovided the disaccharide 21 (0.043 g, 70%). ¹ H-nmr: 5.90(m, 1H,--CH═), 5.53(m, 1H, H-8'), 5.38(dd, 1H, J_(6'),7' 2.7 Hz, J_(7'),8' 9.0Hz, H-7'), 5.20(m, 2H incl. PhCH and ═CH), 5.08[m, 2H, incl. H-2(dd,J₁,2 8.0 Hz, J₂,3 10.0 Hz)], 4.83(ddd, 1H, J_(3'e),4' 4.5 Hz, J_(3'a),4'13.5 Hz, J_(4'),5' 10.0 Hz, H-4'), 4.53(d, 1H, H-1), 2.70(dd, 1H,J_(3'a),3'e 4.5 Hz, H-3'e), 2.18, 2.17, 2.10, 2.08, 2.06, 2.04, 1.88(7s, 22H, 6 OAc, 1 NAc overlapping with H-3'a).

Preparation of allyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-galactopyranosyl-(1-4)-0]-(2,6-di-O-acetyl-β-D-galactopyranoside)(22)

A solution of silver trifluoromethanesulfonate (11.7 mg, 0,0456 mmol) intoluene (0.4 ml) was syringed into a mixture of the disaccharide 21(0.013 g, 0.0152 mmol), molecular sieves 4A (0.070 mg, crushed) and3,4,5-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-galactopyranosyl chloride(0.024 g, 0.053 mmol) in dichloromethane (0.6 ml) at -35° C. The mixturewas stirred for 2.5 hours at -35° C. then one hour at -20° C. Aftercooling at -78° C. methanol was added. The mixture was then brought to0° C. diluted with dichloromethane, filtered and the solventsuccessively washed with a solution of sodium bicarbonate and water.Drying and evaporation left a residue (0.050 g) which waschromatographed on silica gel (2 g) using a mixture of hexane, ethylacetate and ethanol (10:10:1) as eluant. Evaporation of the appropriatefractions gave 22 (0.013 g, 67%). ¹ H-nmr: 5.84[m, 2H, incl. --CH═(m)and H-3"(dd, J_(3"), 4" 3.5 Hz, J_(2"),3" 11.5 Hz)], 5.40(m, 2H incl.H-8' and PhCH), 5.13[m, 2H, incl. H-1"(d, J_(1"),2" 8.0 Hz)], 4.78(ddd,1H, J_(3'e),4' 4.5 Hz, J_(3'a),4' 13.5 Hz, J_(4'),5' 10.0 Hz, H-4'),4.60(dd, 1H, J₁,2 8.0 Hz, J₂,3 10.0 Hz, H-2), 4.50[m, 2H, incl. H-2"(dd)and H-1(d)], 3.02(dd, 1H, J_(3'a),3'e 13.5 Hz, H-3'e), 2.15(two), 2.14,2.10, 2.05, 2.02(two), 1.88, 1.87, 1.86 (8s, 30H, 9 OAc, 1 NAc), 1.40(t,1H, H-3'a).

EXAMPLE VII SYNTHESIS OF TRISACCHARIDES: COMPOUNDS 27 AND 308-Methoxycarbonyloctyl 6-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside(23)

3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-glucopyranosyl bromide (5 g,0.0126 mmol) in dichloromethane (5 ml) was added dropwise in 0.5 hourinto a mixture of 8-methoxycarbonyl octanol (5.0 g), molecular sieves 4A(7.5 g, crushed), dry silver carbonate (4.5 g, 0.053 mmol) indichloromethane (5 ml) stirred and cooled at -20° C. The mixture wasbrought to -10° C. and stirred for 3-4 hours at which time TLC developedwith hexane:ethyl acetate (60:40) indicated that the reaction wascomplete. The mixture was then diluted with dichloromethane, filtered oncelite washed with water (twice). The crude product obtained afterevaporation was dissolved in pyridine (30 ml) and acetylated with aceticanhydride (1.5 ml) at 22° C. for 48 hours. TLC indicated that theunreacted alcohol had been acetylated. Methanol was added to the mixturewhich was then diluted with dichloromethane washed with water, asolution of sodium bicarbonate, water and brine. The crude product waschromatographed on silica gel using a mixture of hexane and ethylacetate (75:25) as the eluant which gave a pure product (5.5 g, 90%).The material was crystallized from ethanol: [α]²² _(D) -13.2°(c1.0,chloroform); m.p. 59°-61°; ¹ H-nmr: 5.00(m, 2H, H-3 and H-4), 4.39(d,1H, J₁,2 7.5 Hz, H-1), 3.45-4.45[m incl. OCH₃ (s, 3.67)], 2.10, 2.05(2s,6H, 2 OAc).

Anal. Calc. for C₂₂ H₃₅ O₁₀ N₃ : C, 52.68; H, 7.03; N, 8.38, Found: C,52.74; H, 6.90; N, 8.42.

A 0.2N solution of sodium methoxide in methanol (0.5 ml) was syringedinto a flask containing the above compound (5.5 g, 0.011 mmol) inmethanol (160 ml). After 1 day at 22° C., some Dowex(H⁺) resin was addedto the solution. After stirring and filtration, evaporation of thesolvent left a residue which was used directly in the next step withoutcharacterization.

Acetyl chloride (0.408 ml, 5.75 mmol) in dichloromethane (12 ml) wasadded dropwise (45 minutes) to a solution of the triol (4.79 mmol) andpyridine (0.462 ml, 5.75 mmol) in dichloromethane (80 ml) cooled to -78°C. After 30 minutes, TLC (ethyl acetate) indicated the completion of thereaction and some methanol was added. The mixture was diluted withdichloromethane and washed with water. The solvents were evaporated invacuo with an excess of toluene and the residue chromatographed onsilica gel (100 g) using a mixture of hexane:ethyl acetate (45:55). Pure23 (1.71 g, 89%) was obtained as a syrup, [α]²² _(D) -24°(c1.0,chloroform); ¹ H-nmr: 4.33(d, J₁,2 7.5 Hz, H-1), 3.68(s, 3H, OCH₃),2.15(s, 3H, OAc).

Anal. Calc. for C₁₈ H₃₁ O₈ N₃ : C, 51.78; H 7.49; N, 10.07. Found: C,51.42; H, 7.89; N, 10.56.

8-Methoxycarbonyloctyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-3)-O-(6-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside)(24) and 8-methoxycarbonyloctyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-O-(6-O-acetyl-2-azido-2-deoxy-β-D-glucopyranoside)(25)

Trimethylsilyl trifuoromethanesulfonate (0.159 ml, 0.183 g, 0.813 mmol)in dichloromethane (2 ml) was added portionwise (1 hour) to a mixture of15 (0.730 g, 0.813 mmol), the diol 23 (0.649 g, 1.62 mmol) and drierite(0.650 g, crushed) in dichloromethane (5 ml) at 22° C. TLC (hexane:ethyl acetate:ethanol 10:10:1) indicated that the reaction progressedrapidly after more than 0.5 equivalent of the trimethylsilyl triflatehad been added. The reaction was quenched by addition of triethylamineafter one hour. The mixture was diluted with dichloromethane, filteredand washed with a solution of sodium bicarbonate, water and brine.Evaporation and drying in vacuo left a residue (1.5 g).

The crude product (4.70 g) obtained from 15 (2.63 g, 2.95 mmol) waschromatographed on silica gel (140 g) using a mixture of hexane:acetone(4:6) as eluant. This separated the unreacted starting 23 (0.020 g) andother compounds from a 2:1 mixture of the two trisaccharides 24 and 25(2.60 g, 70%). Further column chromatography on TLC grade silica geleluted with a mixture of hexane:ethyl acetate:ethanol (30:70:1) underpressure gave the trisaccharides 24 (1.516 g) and 25 (0.794 g).Trisaccharide 24: foam, [α]²² _(D) +0.188°(c1.0 chloroform); i.r. 2115cm⁻¹ (N₃); ¹ H-nmr: 7.400(m, 5H, aromatics), 5.538(ddd, 1H, J_(7"),8"9.0 Hz, J_(8"),9"a 2.7 Hz, J_(8"),9"b 5.2 Hz, H-8"), 5.450(d, 1H,J_(gem) 12.5 Hz, PhCH), 5.350(dd, 1H, J_(6"),7" 2.7 Hz, H-7"), 5.095(dd,1H, J_(1'),2' 8.0 Hz, J_(2'),3' 10.0 Hz, H-2'), 5.055(d, 1H, J_(gem)12.5 Hz, PhCH), 5.000(bd, 1H, J_(3'),4' 3.5 Hz, H-4'), 4.875[m, 2H incl.NH(J_(5"),NH 10.0 Hz) and H-4")], 4.693[m, 2H incl. H-1'(d) andH-3'(dd)], 4.275[m, 2H incl. H-1(J₁,2 7.5 Hz)], 3.662(s, 3H, OCH₃),3.325(dd, 1H, J₂,3 10.0 Hz, H-2), 3.260(dd, 1H, J₃,4 9.0 Hz H-3),2.615(dd, 1H, J_(3"a),4" 4.5 Hz, J_(3"a),3"e 13.5 Hz, H-3"e), 2.245,2.188, 2.085(two), 2.070(two), 2.065, 1.988, 1.713(9 s, 27H, 8 OAc, 1NAc), 1.588(t, 1H, J_(3"a),4" 13.0 Hz H-3"a).

Anal. Calc. for C₅₆ H₇₈ O₂₈ N₄ : C, 53.53; H, 6.26; N, 4.46. Found: C,53.27; H, 6.27; N, 4.50.

Trisaccharide 25: foam [α]²² _(D) +0.240°(c1.0 chloroform): i.r. 2113cm⁻¹ (N₃); ¹ H-nmr: 7.400(m, 5H, aromatics), 5.513(ddd, J_(7"),8" 8.5Hz, J_(8"),9"a 2.6 Hz, J_(8"),9"b 5.2 Hz, H-8"), 5.438(d, 1H, J_(gem)12.0 Hz, PhCH), 5.339(dd, 1H, J_(6"),7" 2.7 Hz, H-7"), 5.063[m, 3H incl.PhCH(d), NH(d, J_(5"),NH 10.5 Hz, H-2'(dd, J_(1'),2' 8.0 Hz, J_(2'),3'10.0 Hz)], 4.988(bd, 1H, J_(3'),4' 3.5 Hz, H-4'), 4.867(ddd, 1H,J_(3"e),4" 4.5 Hz, J_(3"a),4" 12.5 Hz, J_(4"),5" 10.5 Hz, H-4"),4.638[m, 2H incl. H-1'(d) and H-3'(dd)], 4.238[m, 2H, incl. H-1(d J₁,28.0 Hz)], 3.662(s, 3H, OCH₃), 3.330(dd, 1H, J₂,3 10.0 Hz, H-2),2.663(dd, 1H, J_(3"a),3"e 12.5 Hz, H-3"e), 2.255, 2.168, 2.138,2.075(three), 2.055, 1.988, 1.838(7 s, 27H, 8 OAc, 1 NAc), 1.663(t, 1H,H-3"a).

Anal. Calc. for C₅₆ H₇₈ O₂₈ N₄ : C, 53.53; H, 6.26; N, 4.46. Found: C,53.41; H, 6.30; N, 4.73.

For identification purposes both trisaccharides 24 and 25 wereacetylated (pyridine, acetic anhydride and DMAP). After the usual workup the recovered products were filtered through silica gel using ethylacetate as eluant. The appropriate fractions were pooled and evaporated.Decoupling experiments on the ¹ H-nmr spectra confirmed the structuresof both compounds.

Trisaccharide 26, ¹ H-nmr: 7.43(m, 5H, aromatics), 5.538(ddd, J_(7"),8"8.5 Hz, J_(8"),9"a 2.7 Hz, J_(8"),9"b 5.6 Hz, H-8"), 5.075(d, 1H,J_(gem) 12.0 Hz, PhCH), 5.355(dd, 1H, J_(6"), 7" 2.5 Hz, J_(7"),8" 8.5Hz, H-7"), 4.85-5.05[m, 6H incl. H-4'(d, J_(3'),4' 3.5 Hz), H-2'(dd,J_(1'),2' 8.0 Hz, J_(2'),3' 10.0 Hz), H-1'(d), H-4, H-4"(m)], 4.650(dd,1H, H-3'), 4.280(d, 1H, J₁,2 8.0 Hz, H-1), 3.662(s, OCH₃), 3.638(t, 1H,J₂,3 =J₃,4 9.5 Hz, H-3), 3.363(dd, 1H, H-2), 2.613(dd, 1H, J_(3"e),4"5.0 Hz, J_(3"e),3"a 13.0 Hz, H-3"e), 2.250, 2.180, 2.075(three),2.050(two), 2.030, 1.975, 1.825(7 s, 30H, 9 OAc, 1 NAc), 1.688(t,J_(3"a),4" 12.5 Hz, H-3"a).

Trisaccharide 28, ¹ H-nmr: 7.40(m, 5H aromatics), 5.450[m, 2H, incl.H-8"(m) and PhCH(d, J_(gem) 12.0 Hz)], 5.368(dd, 1H, J_(6"),7" 2.5 Hz,J_(7"),8" 8.5 Hz, H-7"), 5.050(d, 1H, PhCH), 5.000(bd, 1H, J_(3'),4' 3.5Hz, H-4'), 4.80-4.98[m, 4H incl. H-3(dd, J₂,3 10.0 Hz, J₃,4 9.5 Hz),H-2'(dd, J_(1'),2' 8.0 Hz, J_(2'),3' 10.0 Hz), NH(d) and H-4"(m)],4.588(d, 1H, H-1'), 4.550(dd, 1H, H-3'), 4.337(d, 1H, J₁,2 8.5 Hz, H-1),3.750(t, 1H, J₄,5 10.0 Hz, H-4), 3.638(s, 3H, OCH₃), 3.387(dd, 1H, H-2),2.590(dd, 1H, J_(3"e),4" 5.0 Hz, J_(3"e),3"a 13.0 Hz, H-3"e), 2.207,2.160, 2.108(two) 2.090, 2.070, 2.055, 2.027, 1.977, 1.825,(9 s, 30H, 9OAc, 1 NAc) 1.665(t, J_(3"a),4" 12.5 Hz, H-3"a).

8-Methoxycarbonyloctyl(5-acetamido-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)-O-(β-D-galactopyranosyl)-(1-3)-O-(2-acetamido-2-deoxy-.beta.-D-glucopyranoside)(27)

Reduction of the azido group of compound 24 (0.100, 0.796 mmol) withhydrogen sulfide in a mixture of pyridine (3 ml), water (0.5 ml) andtriethylamine (0.035 ml), followed by N-acetylation with aceticanhydride afforded the 2-acetamidotrisaccharide (76%). Hydrogenation ofthis compound (0.136 g, 0.104 mmol) in the presence of palladium oncarbon and subsequent de-O-acetylation gave the title trisaccharide 27(0.070 g, 79.5%), [α]²² _(D) -19.8°(c1.0, water); ¹ H-nmr(D₂ O):4.555(d, 1H, J 8.2 Hz) and 4.490(d, 1H, J 8.0 Hz): H-1 and H-1',4.083(dd, 1H, J_(2'),3' 10.0 Hz, J_(3'),4' 3.2 Hz, H-3'), 3.683(s,OCH₃), 2.763(dd, 1H, J_(3"e),4" 4.6 Hz, J_(3"e),3"a 12.1 Hz, H-3"e),2.388(t, 2 H, J 6.5 Hz, CH₂ CO), 2.025, 2.015(2 s, 6H, 2 NAc), 1.788(t,1H, J_(3"a),4" 12.0 Hz, H-3"a), 1.61[m, 4H, (CH₂)₂ ], 1.363[m, 8H,(CH₂)₄ [.

8-Methoxylcarbonyloctyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1-4)-O-(2-acetamido-6-O-acetyl-2-deoxy-β-D-glucopyranoside)(29)

The azido group of compound 25 (0.410 g, 0.327 mmol) was reduced asindicated above for the preparation of compound 27. Chromatographyprovided compound 29 (0.326 g, 78.5%) as a syrup: [α]²² _(D)+21.2°(c1.0, chloroform); ¹ H-nmr: 7.40(m, 5H, aromatics), 5.575(d, 1H,J₂,NH 8.0 Hz, NH-2), 5.475(ddd, 1H, J_(7"),8" 8.5 Hz, J_(8"),9"a 5.6 Hz,J_(8"),9"b 2.6 Hz, H-8"), 5.440(d, 1H, J_(gem) 12.5 Hz, PhCH), 5.350(dd,1H, J_(6"),7" 2.6 Hz, H-7"), 5.050[m, 2H incl. H-2'(dd, J_(1'),2' 8.0Hz, J_(2'),3' 10.0 Hz and PhCH (d)], 5.000(bd, 1H, J_(3'),4' 3.5 Hz,H-4' ), 4.875[m, 2H, incl. NH-5"(d, J_(5"),NH 10.0 Hz) and H-4"(m)],4.775(d, 1H, J₁,2 8.0 Hz, H-1), 4.650[m, 2H, incl. H-1'(d) andH-3'(dd)], 3.675(s, 3H, OCH₃), 3.500[m, 3H, incl. H-6"(dd, J_(5"),6"10.5 Hz) and H-2(m)], 2.600(dd, 1H, J_(3"a),4" 4.0 Hz, J_(3"a),3"e 13.0Hz, H-3"e), 2.262, 2.168, 2.082, 2.075(three), 2.050, 1.980(two),1.825(7 s, 30H, 8 OAc, 2 NAc).

Anal. Calc. for C₅₈ H₈₂ O₂₉ N₂ : C, 54.80; H, 6.50; N, 2.20. Found: C,54.51; H, 6.54; N, 2.50.

The i.r. spectrum showed the absence of the azide absorption.

8-Methoxycarbonyloctyl(5-acetamido-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)-O-(β-D-galactopyranosyl)-(1-4)-O-(2-acetamido-2-deoxy-.beta.-D-glucopyranoside)(30)

The trisaccharide 29 was deprotected and purified as indicatedpreviously for the preparation of compound 27 to provide for compound 30(79%): [α]²² _(D) -8.3°(c 1.0, water); ¹ H-nmr(D₂ O): 4.550(d, 1H, J 8.0Hz) and 4.513(d, 1H, J 7.2 Hz): H-1 and H-1', 4.115(dd, 1H, J_(2'),3'10.0 Hz, J_(3'),4' 3.0 Hz, H-3'), 3.955(bd, 1H, H-4'), 3.685(s, OCH₃),2.755(dd, 1H, J_(3"e),3"a 12.5 Hz, J_(3"e),4" 4.6 Hz, H-3"e), 2.388(t,2H, J 6.5 Hz, CH₂ CO), 2.025(s, 6H, 2 NAc), 1.800(t, 1H, J_(3"a),4" 12.5Hz, H-3"a), 1.600[m, 4H, (CH₂ )₂ ], 1.325[m, 8H, (CH₂)₄ ].

EXAMPLE VIII Preparation of 8-methoxycarbonyloctyl (benzyl5-acetamido-4,7,8,9,-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(4,6-di-O-acetyl-2-O-benzoyl-β-D-galactopyranosyl)-(1-4)-O-(2-ezido-2-deoxy-β-D-glucopyranoside)(32) and of 8-methoxycarbonyloctyl (benzyl5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)-O-(4,-di-O-acetyl-2-O-benzoyl-glucopyranosyl)-(1-3)-O-(2-azido-2-deoxy-β-D-glucopyranoside)(33)

Trimethysilyltrifluoromethane sulfonate (0.159 g, 0.715 mmol) indichloromethane (1.5 mL) was syringed in three portions (every hour)into the a flask containing 16 (0.335 g, 0.355 mmol), calcium sulfate(0.500 g, crushed), the diol 31 (0.435 g, 0.710 mmol prepared from theazido glucoside used in Example VII by selective silylation) indichloromethane (3 mL). After 4 hours at 22° C., the reaction wasstopped by addition of triethylamine (0.071 mL). Dilution withdichloromethane, filtration and successive washing with aqueous sodiumbicarbonate and water gave the crude product after drying andevaporation. Chromatography on silica gel (36 g) using a mixture ofhexane:ethyl acetate:ethanol (10:10:1) gave a mixture (2:1) of the twotrisaccharides with 1-4 and 1-3 linkages, respectively (0.432 g, 81%).

Tetraethylammonium chloride (0.392 g, 2.63 mmol), potassium fluoride(0.153 g, 2.63 mmol) and benzoic acid (0.053 g, 0.434 mmol) were addedto the above mixture (0.0590 g, 0.394 mmol) in acetonitrile (15 mL).After stirring overnight at 22° C., the solvent was evaporated in vacuo,the residue diluted with dichloromethane and washed with water. Dryingand evaporation left the crude mixture (0.574 g) which waschromatographed on silica gel (37 g) using a mixture of chloroform andacetone (70:30). Collection and evaporation of the appropriate fractionsgave 32 (0.360 g, 60%): i.r. 2113 cm⁻¹ (N₃); ¹ H-nmr: 5.78(m, 1H, H-8"),5.46(d, 1H, J_(gem) 12.0, PhCH), 5.33(dd, 1H, J_(1'),2' 8.0 Hz,J_(2'),3' 10.0 Hz, H-2'), 4.18(d, 1H, J₁,2 8.0 Hz, H-1 ), 3.68(s, 3H,OCH₃), 3.30(dd, 1H, J₁,2 8.0 Hz, J₂,3 10.0 Hz H-2), 2.56(dd, J_(3"e),4"4.5 Hz, J_(3"e),3"a 13.0 Hz, H-3"e), 2.28(t, 2H, J 7.5 Hz, CH₂ CO₂),2.22, 2.14, 2.12, 2.08, 1.94, 1.76, 1.52,(7 s, 21H, 6 OAc, 1 NAc),1.68,(t, J_(3"a),4" 13.0 Hz, H-3"a) and compound 33 (0.180 g, 29%): i.r.2113 cm⁻¹ (N₃); ¹ H-nmr: 5.60(m, 1H, H-8"), 5.44(d,1H, J_(gem) 12.0,PhCH), 5.47(dd, 1H, J_(1'),2' 8.0 Hz, J_(2'),3' 10.0 Hz, H-2'), 5.20(dd,1H, J_(6"),7" 2.5 Hz, J_(7"),8" 10.0 Hz, H-7"), 4.28(d, 1H, J₁,2 8.0 Hz,H-1), 3.67(s, 3H, OCH₃), 2.68(dd, 1H, J_(3"e),4" 4.5 Hz, J_(3"e),4" 13.0Hz, H-3"e), 2.28(t, 2H, J 7.5 Hz, CH₂ --CO), 2.22, 2.12, 2.09,(two),1.96, 1.78, 1.58,(6 s, 21H, 6 OAc, 1 NAc), 1.83(t, 1H, J_(3"a),4" 13.0Hz, H-3"a).

For identification purposes, both trisaccharides 32 and 33 wereperacetylated with acetic anhydride, pyridine and dimethylaminopyridine.Peracetylated 32; ¹ H-nmr: 4.89(dd, 1H, J₂,3 10.5 Hz, J₃,4 9.7 Hz, H-3),4.23(d, J₁,2 8.0 Hz, H-1), 3.78(t, J₄,5 10.0 Hz, H-4), 2.37 (dd, H-2).Peracetylated 33, ¹ H-nmr: 4.80(m, 4H, incl. H-4), 4.20 (d, J₁,2 8.0 Hz,H-1), 3.67[m, 4H incl. OCH₃ (s) and H-3], 3.20 (dd, J₂,3 10.0 Hz, H-2).In both spectra the signals, which are overlapping with others, havebeen identified by decoupling experiments.

Preparation of 8-methoxycarbonyloctyl(5-acetamido-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)-O-(β-D-galactopyranosyl)-(1-4)-(2-acetamido-2-deoxy-.beta.-D-glucopyranoside)(30)

Hydrogen sulfide was bubbled through a solution of the trisaccharide 32(0.070 g, 0.055 mmol), triethylamine (0.050 mL, water (0.500 mL) andpyridine (2.00 mL). After 20 hours at 22° C., acetic anhydride (0.250mL) was syringed into the cooled mixture which was then co-evaporatedwith an excess of toluene. Chromatography of the residue on silica gel(7 g) using a mixture of toluene:ethanol (10:1) gave the pureintermediate (0.049 g, 68%). ¹ H-nmr: 5.66(m, 1H, H-8"), 5.37(d, 1H,J_(gem) 12.0 Hz, PhCH), 5.25(dd 1H, J_(1'),2' 8.0 Hz, J_(2'),3' 10.0 Hz,H-2'), 3.59[m, incl. OCH₃ (s)], 2.47(dd, 1H, J_(3"e),3"a 13.5 Hz,J_(3"e),4" 4.5 Hz, H-3"e), 2.14, 2.04, 2.03, 2.02, 1.92, 1.86, 1.68,1.43,(8 s, 24H, 6 OAc, 2 NAc). i.r. showed the absence of azideabsorption.

The above intermediate (0.049 g, 0.0375 mmol) was reduced at atmospherepressure in methanol (2 mL) in the presence of Pd/C (5%, 0.050 g) for 2hours. Removal of the catalyst and evaporation left a reside (0.042 g)which was treated with a 0.2N solution of sodium methoxide in methanolfor 3 days at 22° C. Deionization with resin (IRC 50, H+ form),filtration and evaporation left a residue (0.034 g) which waschromatographed on Iatrobeads (6RS 8060, 1.5 g) using a mixture ofchloroform, methanol and water (65:35:8) as eluant giving 30 (0.037 g,75%): [α]²² _(D) -8.3°(c1.0, water); ¹ H-nmr: 4.55(d, 1H, J 8.0 Hz) and4.51(d, 1H, J 7.2 Hz): H-1 and H-1', 4.11(dd, 1H, J_(2'),3' 10.0 Hz,J_(3'),40' 3.0 Hz, H-3'), 3.68(s, OCH₃), 2.75(dd, 1H, J_(3"e),4" 4.5 Hz,J_(3"e),3"a 12.5 Hz, H-3"e), 2.38(t, 2H, J 6.5 Hz, CH₂ CO), 2.025(s, 6H,2 NAc), 1.80(t, 1H, J_(3"a),4" 12.5 Hz, H-3"a), 1.60(m, 4H) and 1.32(m,8H): methylenes.

EXAMPLE IX PREPARATION OF COMPOUND 35: THE 19-9 TETRASACCHARIDE8-Methoxycarbonyloctyl(5-acetamido-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)-O-β-D-galactopyranosyl-(1-3)-O-[α-L-fucopyranolsyl-(1-4)-O-]-2-acetamido-2-deoxy-β-D-glucopyranoside(35)

Tri-O-benzyl fucopyranosyl bromide freshly prepared from tri-O-benzylfucopyranose (1.78 g, 4.11 mmol) in dichloromethane (2 ml) was added tothe starting material 24 (0.840 g, 0.68 mmol), molecular sieves 4A (1.0g, crushed), dry tetraethylammonium bromide (0.144 g, 0.686 mmol) anddimethylformamide (0.50 ml) in dichloromethane (2.0 ml). The mixture wasstirred at 22° C. TLC (chloroform:acetone 70:30 and hexane:ethylacetate:ethanol 10:10:1) indicated a complete reaction in about 30hours. Some methanol was added and stirring was continued for a fewhours. The content of the flask was diluted with dichloromethane,filtered on paper, washed with a solution of sodium bicarbonate, waterand brine. The crude product obtained was chromatographed on silica gel(90 g) using a mixture of hexane:ethyl acetate:ethanol (70:30:1) aseluant, to give compound 34 (0.978 g, 86%) as a syrup: [α]²² _(D)+0.125°(c1.0 chloroform); i.r. 2116 cm⁻¹ (N₃); ¹ H-nmr: 7.30(m, 20H,aromatics), 5.500(m, 1H, J_(7"'),8"' 8.6 Hz, J_(8"'),9"'a 2.8 Hz,J_(8"'),9"'b 4.2 Hz, H-8"'), 5.450(d, 1H, J_(gem) 12.0 Hz, PhCH),5.387(dd, 1H, J_(6"'),7"' 2.8 Hz, H-7"'), 5.325(d, 1H, J_(1'),2' 8.0 Hz,H-1'), 5.313(d, 1H, PhCH), 5.287(bd, 1H, J_(3'),4' 3.5 Hz, H-4'),4.967(dd, 1H, J_(2'),3' 10.0 Hz, H-2'), 4.674(dd, 1H, H-3'), 4.300(dd,1H, J_(9"'a),9"'b 12.5 Hz, H-9"'a), 4.217(d, 1H, J₁,2 8.0 Hz, H-1),3.750(t, 1H, J₃,4 =J₄,5 9.5 Hz, H-4), 3.655(s, 3H, OCH₃), 3.612(t, 1H,J₂,3 9.5 Hz, H-3), 3.495(dd, J_(5"'),6"' 11.0 Hz, H-6"'), 3.250(dd,H-2), 2.587(dd, 1H, J_(3"'e),4"' 4.5 Hz, J_(3"'a),3"'e 13.5 Hz, H-3"'e),2.250, 2.195, 2.060(two), 2.040, 2.030, 1.980, 1.825, 1.755(27H, 8 OAc,1 NAc) 1.662(t, J_(3"'a),4"' 13.5 Hz, H-3"'a), 1.237(d, J_(5"),6" 7.5Hz, H-6").

Anal. Calc. for C₈₃ H₁₀₆ O₃₂ N₄ : C,59.6; H, 6.39; N, 3.35. Found: C,59.36; H, 6.40; N, 3.22.

Hydrogen sulfide was slowly bubbled into a solution of the startingmaterial 34 (0.600 g, 0.359 mmol) in a mixture of pyridine (39 ml),water (5.8 ml) and triethylamine (1.45 ml) while cooling in ice for 2hours and at room temperature for about 5 hours. After overnight at 22°C., TLC (toluene:ethanol 10:1 or chloroform: acetone 85:15) indicatedthe completion of the reaction at which time acetic anhydride (4.5 ml)was added.

From similar glycosylation reactions compound 34 (0.860 g, 0.514 mmol)was obtained and treated as above. The crude material was co-evaporatedwith an excess of toluene. The residue was then applied on a column ofsilica gel (70 g) and eluted with toluene (300 ml) and a mixture oftoluene and ethanol (100:1, 600 ml) removed all colored material.Elution with the same solvents (100:7) gave some minor fractionsfollowed by the main product with the 2-acetamido group (0.750 g, 86%)as a syrup: [α]²² _(D) -12.9°(c1.0 chloroform); ¹ H-nmr: 7.40(m, 20H,aromatics) 6.220(bd, 1H, J₂,NH 10.0 Hz, NH-2), 5.425[m, 2H, incl.PhCH(d, J_(gem) 12.0 Hz) and H-8"'(m)], 5.350(dd, 1H, J_(6"'),7"' 2.5Hz, J_(7"'),8"' 9.0 Hz, H-7"'), 5.075[m, 2H incl. PhCH (d), and H-4'(bd,J_(3'),4' 3.5 Hz)], 4.675-5.025[m, incl. H-2'(dd, J_(1'), 2' 8.0 Hz,J_(2'),3' 10.0 Hz)], 4.563(dd, H-3'), 3.65(s, 3H, OCH₃), 3.450(dd, 1H,J_(5"'),6"' 11.5 Hz, H-6"'), 2.575(dd, 1H, J_(3"'e),3"'a 13.0 Hz,J_(3"'e),4"' 4.5 Hz, H-3"'e), 2.205, 2.168, 2.072, 2.050(three), 1.975,1.850, 1.830, 1.813(30H, 8 OAc, 2 NAc), 1.60[m, 5H, incl. H-3"'a and(CH₂)₂ ], 1.200(d, J_(5"),6" 7.5 Hz, H-6").

Irradiation of NH(d) at 6.22 indicates that H-2 is at 3.80.

The i.r. spectrum indicated the absence of azide absorption.

Anal. Calc. for C₈₅ H₁₁₀ O₃₃ N₂ : C, 60.48; H, 6.57; N, 1.70. Found:C,60.36; H, 6.46; N, 1.70.

This intermediate tetrasaccharide (0.715 g, 0.436 mmol) was hydrogenatedat atmospheric pressure in methanol (60 ml) in the presence of catalyst(5% Pd/C, 0.700 g, pre-hydrogenated in the same solvent and decanted).TLC developed with chloroform:methanol:water (65:35:3) indicated a rapidreaction. After 4 hours, the catalyst was filtered on paper and washedseveral times with methanol. The solvent was evaporated in vacuo. Thisproduct was dissolved in a 0.2N sodium methoxide in methanol (20 ml) andstirred at 22° C. for 3 days. TLC developed withchloroform:methanol:water (65:35:8) was used to monitor the reaction.After complete reaction, the solution was cooled to-10° C. and someresin (IRC 50, H+ form, methanol washed, 6 g) was added portion wiseuntil neutral pH. Evaporation and freeze drying left a slightlyyellowish powder (0.435 g, quantitative) which was further run throughIatrobeads (6RS 8060) using a mixture of chloroform:water:methanol:(65:35:6) to give pure compound 35 :[α]²² _(D) -49.4°(c1.0, chloroform);¹ H-nmr(D₂ O): 5.005(d, 1H, J_(1'),2' 4.0 Hz, H-1"), 4.866(q, 1H,J_(5"),6" 6.8 Hz, H-5"), 4.525(d, 2H, J₁,2 =J_(1'),2' =7.8 Hz, H-1 andH'-1'), 4.056(m, 2H, incl. H-3'), 3.691(s, OCH₃), 2.767(dd, 1H,J_(3"'a),4"' 4.6 Hz, J_(3"'a),3"'e 12.8 Hz, H-3"'e), 2.392(t, 2H, J 6.5Hz, CH₂ --CO), 1.925, 1.979(2 s, 6H, 2 NAc), 1.765(t, 1H, J_(3"'a),4"'12.6 Hz, H-3"'a), 1.584[m, 4H, (CH₂)₂ ], 1.296[m, 8H, (CH₂)₄ ], 1.170(d,3H, H-6").

EXAMPLE X SYNTHESIS OF COMPOUND 37: SIALO-X TETRASACCHARIDE8-Methoxycarbonyloctyl(5-acetamido-3,5-di-deoxy-α-D-glycero-D-galacto-2-nonulopyranosylonicacid)-(2-3)-O-β-D-galactopyranosyl-(1-4)-O-[α-L-fucopyranosyl-(1-3)-O-]-2-acetamido-2-deoxy-β-D-glucopyranoside(37)

The starting material 29 (0.139 g, 0.109, mmol) was reacted withtri-O-benzyl fucopyranosyl bromide as indicated previously for thepreparation of 34. TLC (chloroform:acetone 70:30 and toluene:ethanol100:10) indicated the completion of the reaction in less than 24 hours.Work-up as indicated before and purification of the reaction product bychromatography on silica gel (8 g) using a mixture of chloroform:acetone(75:25) gave 36 (0.143 g, 77%) as a syrup: [α]²² _(D) -12.0°(c1.0,chloroform); ¹ H-nmr: 7.35(m, 25H aromatics), 5.870(d, 1H, J₂,NH 8.0 Hz,NH-2), 4.975(m, 1H, H-8"'), 4.925(d, 1H, J_(gem) 12.5 Hz, PhCH),5.375(dd, 1H, J_(6"'),7"' 2.7 Hz, J_(7"'),8"' 9.0 Hz, H-7"'), 4.413(q,1H, J_(5'),6' 7.0 Hz, H-5'), 3.67(s, OCH₃), 3.487[m, 2H, incl. H-6"'(dd,J_(5"'),6"' 10.0 Hz)], 2.587(dd, 1H, J_(3"'e),4"' 4.5 Hz, J_(3"'a),3"'e13.0 Hz, H-3"'e), 2.200, 2.163, 2.063(two), 2.038(two), 1.975, 1.875,1.825, 1.750(8 s, 30H, 8 OAc, 2 NAc), 1.18(d, 3H, H-6').

The tetrasaccharide 36 (0.274 g, 0.166 mmol) was de-O-protected aspreviously indicated for the preparation of 35. The crude materialrecovered after de-O-acetylation (0.140 g) was chromatographed onIatrobeads (3 g) using a mixture of chloroform: methanol:water (65:35:5)which provided compound 37 (0.118 g, 74%): [α]²² _(D) -41.0°(c1.0,water); ¹ H-nmr(D₂ O): 5.100(d, 1H, J_(1'),2' 3.8 Hz, H-1'), 4.825(q,1H, J_(5'),6' 6.5 Hz, H-5'), 4.520(d, 2H, J₁,2 =J_(1"),2" 8.0 Hz, H-1and H-1"), 4.085(dd, 1H J_(3"),4" 4.0 Hz, J_(2"),3" 9.8 Hz, H-3")3.668(s, OCH₃), 2.763(dd, 1H, J_(3"'e),4"' 4.6 Hz, J_(3"'e),3"' a 12.4Hz, H-3"'e), 2.388(t, 2H, J 7.5 Hz, CH₂ CO), 2.030, 2.018(2 s, 6H, 2NAc), 1.795(t, 1H, J_(3"'a),4"' 12.2 Hz, 4-3"'a), 1.587[m, 4H, (CH₂)₂ ],1.295[m, 8H, (CH₂)₄ ], 1.165(d, 3H, H-6').

EXAMPLE XI PREPARATION SYNTHETIC ANTIGENS FROM TETRASACCHARIDES 35 AND37

The tetrasaccharide 35 (0.021 g) was treated with 95% hydrazine hydratefor 1 hour to effect the conversion of the methyl ester to the hydrazidederivative compound 38. This product was co-evaporated withn-butanol:water 1:1 several times to effect the removal of residualhydrazine and reacted as follows to give the synthetic antigen.

Compound 38 (0.020 g, 0.020 mmol) was dissolved in dimethylformamide(0.5 ml) and cooled to -20° C. A solution of dioxane (0.020 ml) that was4.0M in hydrochloric acid was added followed by t-butyl nitrite (0.005ml) and the resulting mixture stirred for 30 minutes. At that timesulfamic acid (0.001 g, 0.010 mmol) was added and stirred for 15minutes. This solution was added to a solution of bovine serum albumin(BSA)(0.025 g) in N-ethyldiethanolamine buffer (0.2M, adjusted to pH 9with hydrochloric acid) at 0° C. After standing for 18 hours, thesolution was dialyzed against water for five exchanges with a 10,000molecular weight cut-off. Lyophilization of the contents of the dialysiscell gave the 19-9 synthetic glycoconjugate 43 (0.028 g). Analysis forhexoses by phenol-sulfuric assay indicated the presence of 20 moles ofhapten per mole of BSA. Analysis for N-acetyl neuraminic acidcorroborated this result.

Conversion of the ester 37 to its corresponding hydrazide 39 andreaction of this as described above gave the sialyl-X syntheticglycoconjugate. Similar synthetic glycoconjugates have been preparedwith alternate carrier molecules such as human serum albumin, keyholelimpet hemocyanin and horse radish peroxidase through the reaction ofcompound 38 and 39 as described above.

These products can be used to study the binding properties ofantibodies, bacterial and viral receptors and other biomolecules.

EXAMPLE XII PREPARATION OF SYNTHETIC IMMUNOADSORBENTS FROM COMPOUNDS 38AND 39

The hydrazide 38 or 39 (0.020 g) was converted to the reactive acylazide as described above in example XI and reacted with silylaminatedcrystobilite (20 g) suspended in dry acetonitrile (60 ml) for 18 hoursat which time the solid was filtered and washed with water and thenmethanol. This was then dried at 70° C. to give the syntheticimmunoadsorbent having the reactivity conferred by structure 38 or 39.Phenol-sulfuric and sialic acid assays showed an incorporation of0.7-0.8 micromoles of hapten per gram of support. Many other aminatedsupports have been used to prepare such immunoadsorbents such ascontrolled pore glass, aminated polysaccharides and aminated polymers.These products can be used to isolate, purify or remove antibodies,lectins and other biomolecules which have reactivity or specificity forthe structures of compounds 38 or 39.

EXAMPLE XIII DETECTION OF 19-9 REACTIVE ANTIBODIES WITH THE SYNTHETICGLYCOCONJUGATE PREPARED FROM COMPOUND 38

The wells of plastic plates were coated with the 19-9 syntheticglycoconjugate formed from compound 38 in the following manner. Asolution of the conjugate (50 μg/ml) in buffer (50 mM NaH₂ PO₄ /Na₂HPO₄, 5 mM MgCL₂, 15 mM NaN₃, pH 7.5) (100 μl) was dispensed into eachwell and incubated for 18 hours at ambient temperature at which time thecoating solution was removed by aspiration. A phosphate buffered saline(PBS) solution 5% in BSA (200 μl) was then dispensed into the wells andincubated for 4 hours at ambient temperature at which time this wasremoved by aspiration. The wells were washed successively with 2 times200 μl of PBS and 200 μl of distilled water.

A working solution of antibody was prepared for reaction with the coatedwells in the following manner. With ascites stock, dilution of theantibody with 1% BSA in PBS between 1/50 to 1/100 was done. With cellsupernatant containing antibodies, neat to 1/5 dilutions was used forreaction and with purified antibodies at concentrations in the range of1 mg/ml, dilutions of 1/100 to 1/200 was used. These are only suggesteddilution ranges and these may be altered to suit the purpose of theassay and the nature of the antibody avidities and affinities.

A solution of antibody (100 μl) was dispensed into wells coated with thesynthetic glycoconjugate formed from compound 38 and control wellscoated with other synthetic glycoconjugates, for example, the Lewis^(a)antigen 40, the 2-6 analogue of 38, antigen 41 and the linear 2-3antigen 42. The antibody solution was incubated for 1 to 4 hours andthen removed and the wells were washed with 200 μl of PBS three times.Alkaline phosphatase labelled anti-immunoglobulin in 1% PBS (100 μl) wasthen dispensed into the wells and incubated for 1-3 hours at which timethe wells were aspirated and washed 3 times with PBS. A solution ofphosphatase substrate (100 μl) was then added to the wells and incubatedto allow colour development. The wells were read at A₄ 05 at intervalsto give the data of reactivity of the antibody with the varioussynthetic glycoconjugates as shown below.

    ______________________________________                                        Reaction of Anti-19-9 Antibody with Synthetic Antigens                        ______________________________________                                        Wells Coated With                                                                         40      41      42    43    BSA                                   Absorbance A.sub.405                                                                      0.232   0.148   0.185 0.640 0.201                                 ______________________________________                                    

The above results clearly show specific reaction of this antibody withthe synthetic glycoconjugate 43 formed from the synthetic structure 35.The related conjugates show the same reactivity as the control wellswhich were coated with the BSA carrier molecule. Competitive inhibitionELISA assays were also conducted wherein the free syntheticglycoconjugates were added as inhibitors. Only 43 gave any significantinhibition and this reduced the absorbance to background values. Thisshows that this assay format with the synthetic antigen coated on wellswill function not only as a method for the detection of anti-19-9antibodies but also for the detection of the 19-9 structure itself influids.

We claim:
 1. A compound of the formula: ##STR3## wherein Ac is an acylgroup of from 1 to 6 carbon atoms and Y is selected from the groupconsisting of hydrogen, lower alkyl of from 1 to 6 carbon atoms, alinking arm, a moiety comprising a label, and a moiety comprising achromatographic support.
 2. The compound of claim 1 wherein Y is alinking arm.
 3. The compound of claim 2 wherein the linking arm has theformula: ##STR4## wherein X is a straight or branched chain saturated orunsaturated hydrocarbylene of from 3 to 19 carbon atoms wherein 1 to 3nonadjacent CH₂ units may optionally be replaced by NR, S, or O, whereinR is H or alkyl of from 1 to 6 carbon atoms, and L is a leaving group ora group convertible to a leaving group.
 4. The compound of claim 1wherein Y is lower alkyl of from 1 to 6 carbon atoms.
 5. The compound ofclaim 1 wherein Y comprises a label.
 6. The compound of claim 5 whereinthe label is selected from the group consisting of a radiolabel, afluorophore and an enzyme.
 7. The compound of claim 1 wherein Ycomprises a chromatographic support covalently attached to saidcompound.
 8. The compound of claim 7 wherein the covalent attachment ismade through a linking arm.
 9. The compound of claim 8 wherein thecovalent attachment through the linking arm to the chromatographicsupport is made by reacting the compound having a linking arm of theformula: ##STR5## with the chromatographic support so as to form acovalent attachment wherein X is a straight or branched chain saturatedor unsaturated hydrocarbylene of from 3 to 19 carbon atoms wherein 1 to3 nonadjacent CH₂ units may optionally be replaced by NR, S, or O,wherein R is H or alkyl of from 1 to 6 carbon atoms, and L is a leavinggroup or a group convertible to a leaving group.